Binding domain-immunoglobulin fusion proteins

ABSTRACT

The invention relates to novel binding domain-immunoglobulin fusion proteins that feature a binding domain for a cognate structure such as an antigen, a counterreceptor or the like, a wild-type IgG1, IGA or IgE hinge region polypeptide or a mutant IgG1 hinge region polypeptide having either zero, one or two cysteine residues, and immunoglobulin CH2 and CH3 domains, and that are capable of ADCC and/or CDC while occurring predominantly as polypeptides that are compromised in their ability to form disulfide-linked multimers. The fusion proteins can be recombinantly produced at high express levels. Also provided are related compositions and methods, including cell surface forms of the fusion proteins and immunotherapeutic applications of the fusion proteins and of polynucleotides encoding such fusion proteins.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. application Ser.No. 10/207,655, filed Jul. 25, 2002, now allowed; which is aContinuation-in-Part of U.S. application Ser. No. 10/053,530, filed Jan.17, 2002 (now abandoned); which claims the benefit under U.S.C. §119(e)of U.S. Provisional Application No. 60/367,358, filed Jan. 17, 2001;U.S. application Ser. No. 10/207,655 also claims the benefit underU.S.C. §119(e) of U.S. Provisional Application No. 60/385,691 filed onJun. 3, 2002. All of the above applications are incorporated herein byreference in their entireties.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 910180_(—)401D1_SEQUENCE_LISTING.txt. The textfile is 1393 KB, was created on Feb. 25, 2010, and is being submittedelectronically via EFS-Web, concurrent with the filing of thespecification.

BACKGROUND

1. Technical Field

The present invention relates generally to immunologically active,recombinant binding proteins, and in particular, to molecularlyengineered binding domain-immunoglobulin fusion proteins, includingsingle chain Fv-immunoglobulin fusion proteins. The present inventionalso relates to compositions and methods for treating malignantconditions and B-cell disorders, including diseases characterized byautoantibody production.

2. Description of the Related Art

An immunoglobulin molecule is a multimeric protein composed of twoidentical light chain polypeptides and two identical heavy chainpolypeptides (H₂L₂) that are joined into a macromolecular complex byinterchain disulfide bonds. Intrachain disulfide bonds join differentareas of the same polypeptide chain, which results in the formation ofloops that, along with adjacent amino acids, constitute theimmunoglobulin domains. At the amino-terminal portion, each light chainand each heavy chain has a single variable region that showsconsiderable variation in amino acid composition from one antibody toanother. The light chain variable region, V_(L), associates with thevariable region of a heavy chain, V_(H), to form the antigen bindingsite of the immunoglobulin, Fv. Light chains have a single constantregion domain and heavy chains have several constant region domains.Classes IgG, IgA, and IgD have three constant region domains, which aredesignated CH1, CH2, and CH3, and the IgM and IgE classes have fourconstant region domains, CH1, CH2, CH3 and CH4. Immunoglobulin structureand function are reviewed, for example, in Harlow et al., Eds.,Antibodies: A Laboratory Manual, Chapter 14, Cold Spring HarborLaboratory, Cold Spring Harbor (1988).

The heavy chains of immunoglobulins can be divided into three functionalregions: Fd (fragment comprising VH and CH1), hinge, and Fc (fragmentcrystallizable, derived from constant regions). The Fd region comprisesthe V_(H) and CH1 domains and in combination with the light chain formsFab, the antigen-binding fragment. The Fc fragment is generallyconsidered responsible for the effector functions of an immunoglobulin,such as complement fixation and binding to Fc receptors. The hingeregion, found in IgG, IgA, and IgD classes, acts as a flexible spacer,allowing the Fab portion to move freely in space. In contrast to theconstant regions, the hinge domains are structurally diverse, varying inboth sequence and length among immunoglobulin classes and subclasses.For example, three human IgG subclasses, IgG1, IgG2, and IgG4, havehinge regions of 12-15 amino acids, while IgG3-derived hinge regions cancomprise approximately 62 amino acids, including around 21 prolineresidues and around 11 cysteine residues.

According to crystallographic studies, the immunoglobulin hinge regioncan be further subdivided functionally into three regions: the upperhinge, the core, and the lower hinge (Shin et al., Immunological Reviews130:87 (1992)). The upper hinge includes amino acids from the carboxylend of CH1 to the first residue in the hinge that restricts motion,generally the first cysteine residue that forms an interchain disulfidebond between the two heavy chains. The length of the upper hinge regioncorrelates with the segmental flexibility of the antibody. The corehinge region contains the inter-heavy chain disulfide bridges, and thelower hinge region joins the amino terminal end of the CH2 domain andincludes residues in CH2. (Id.) The core hinge region of human IgG1contains the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 40) which, whendimerized by disulfide bond formation, results in a cyclic octapeptidebelieved to act as a pivot, thus conferring flexibility. The hingeregion may also contain one or more glycosylation sites, which include anumber of structurally distinct types of sites for carbohydrateattachment. For example, IgA1 contains five glycosylation sites within a17 amino acid segment of the hinge region, conferring exceptionalresistance of the hinge region polypeptide to intestinal proteases,considered an advantageous property for a secretory immunoglobulin.

Conformational changes permitted by the structure and flexibility of theimmunoglobulin hinge region polypeptide sequence may affect the effectorfunctions of the Fc portion of the antibody. Three general categories ofeffector functions associated with the Fc region include (1) activationof the classical complement cascade, (2) interaction with effectorcells, and (3) compartmentalization of immunoglobulins. The differenthuman IgG subclasses vary in the relative efficacies with which they fixcomplement, or activate and amplify the steps of the complement cascade(e.g., Kirschfink, 2001 Immunol. Rev. 180:177; Chakraborti et al., 2000Cell Signal 12:607; Kohl et al., 1999 Mol. Immunol. 36:893; Marsh etal., 1999 Curr. Opin. Nephrol. Hypertens. 8:557; Speth et al., 1999 WienKlin. Wochenschr. 111:378). Complement-dependent cytotoxicity (CDC) isbelieved to be a significant mechanism for clearance of specific targetcells such as tumor cells. In general, IgG1 and IgG3 most effectivelyfix complement, IgG2 is less effective, and IgG4 does not activatecomplement. Complement activation is initiated by binding of C1q, asubunit of the first component C1 in the cascade, to an antigen-antibodycomplex. Even though the binding site for C1q is located in the CH2domain of the antibody, the hinge region influences the ability of theantibody to activate the cascade. For example, recombinantimmunoglobulins lacking a hinge region are unable to activatecomplement. (Shin et al., 1992) Without the flexibility conferred by thehinge region, the Fab portion of the antibody bound to the antigen maynot be able to adopt the conformation required to permit C1q to bind toCH2. (See id.) Hinge length and segmental flexibility have beencorrelated with complement activation; however, the correlation is notabsolute. Human IgG3 molecules with altered hinge regions that are asrigid as IgG4 can still effectively activate the cascade.

The absence of a hinge region, or a lack of a functional hinge region,can also affect the ability of certain human IgG immunoglobulins to bindFc receptors on immune effector cells. Binding of an immunoglobulin toan Fc receptor facilitates antibody-dependent cell-mediated cytotoxicity(ADCC), which is presumed to be an important mechanism for theelimination of tumor cells. The human IgG Fc receptor (FcR) family isdivided into three groups, FcγRI (CD64), which is capable of binding IgGwith high affinity, and FcγRII (CD32) and FcγRIII (CD16), both of whichare low affinity receptors. The molecular interaction between each ofthe three receptors and an immunoglobulin has not been definedprecisely, but experimental evidence indicates that residues in thehinge proximal region of the CH2 domain are important to the specificityof the interaction between the antibody and the Fc receptor. Inaddition, IgG1 myeloma proteins and recombinant IgG3 chimeric antibodiesthat lack a hinge region are unable to bind FcγRI, likely becauseaccessibility to CH2 is decreased. (Shin et al., Intern. Rev. Immunol.10:177, 178-79 (1993).)

Unusual and apparently evolutionarily unrelated exceptions to the H₂L₂structure of conventional antibodies occur in some isotypes of theimmunoglobulins found in camelids (Hamers-Casterman et al., 1993 Nature363:446; Nguyen et al., 1998 J. Mol. Biol 275:413) and in nurse sharks(Roux et al., 1998 Proc. Nat. Acad. Sci. USA 95:11804). These antibodiesform their antigen-binding pocket using the heavy chain variable regionalone. In both species, these variable regions often contain an extendedthird complementarity determining region (CDR3) to help compensate forthe lack of a light chain variable region, and there are frequentdisulfide bonds between CDR regions to help stabilize the binding site[Muyldermans et al., 1994 Prot. Engineer. 7:1129; Roux et al., 1998].However, the function of the heavy chain-only antibodies is unknown, andthe evolutionary pressure leading to their formation has not beenidentified. Since camelids, including camels, llamas, and alpacas, alsoexpress conventional H₂L₂ antibodies, the heavy chain-only antibodies donot appear to be present in these animals simply as an alternativeantibody structure.

Variable regions (V_(H)H) of the camelid heavy chain-onlyimmunoglobulins contain amino acid substitutions at several positionsoutside of the CDR regions when compared with conventional (H₂L₂) heavychain variable regions. These amino acid substitutions are encoded inthe germ line [Nguyen et al., 1998 J. Mol. Biol 275:413] and are locatedat residues that normally form the hydrophobic interface betweenconventional V_(H) and V_(L) domains [Muyldermans et al., 1994 Prot.Engineer. 7:1129]. Camelid V_(H)H recombine with IgG2 and IgG3 constantregions that contain hinge, CH2, and CH3 domains but which lack a CH1domain [Hamers-Casterman et al., 1993 Nature 363:446]. Interestingly,V_(H)H are encoded by a chromosomal locus distinct from the V_(H) locus[Nguyen, 1998], indicating that camelid B cells have evolved complexmechanisms of antigen recognition and differentiation. Thus, forexample, llama IgG1 is a conventional (H₂L₂) antibody isotype in whichV_(H) recombines with a constant region that contains hinge, CH1, CH2and CH3 domains, whereas the llama IgG2 and IgG3 are heavy chain-onlyisotypes that lack CH1 domains and that contain no light chains.

Monoclonal antibody technology and genetic engineering methods have ledto rapid development of immunoglobulin molecules for diagnosis andtreatment of human diseases. Protein engineering has been applied toimprove the affinity of an antibody for its cognate antigen, to diminishproblems related to immunogenicity of administered recombinantpolypeptides, and to alter antibody effector functions. The domainstructure of immunoglobulins is amenable to recombinant engineering, inthat the antigen binding domains and the domains conferring effectorfunctions may be exchanged between immunoglobulin classes (e.g., IgG,IgA, IgE) and subclasses (e.g., IgG1, IgG2, IgG3, etc.).

In addition, smaller immunoglobulin molecules have been constructed toovercome problems associated with whole immunoglobulin therapy. Forinstance, single chain immunoglobulin variable region fragmentpolypeptides (scFv) comprise an immunoglobulin heavy chain variabledomain joined via a short linker peptide to an immunoglobulin lightchain variable domain (Huston et al. Proc. Natl. Acad. Sci. USA, 85:5879-83, 1988). Because of the small size of scFv molecules, theyexhibit very rapid clearance from plasma and tissues and are capable ofmore effective penetration into tissues than whole immunoglobulins.(see, e.g., Jain, 1990 Cancer Res. 50:814s-819s.) An anti-tumor scFvshowed more rapid tumor penetration and more even distribution throughthe tumor mass than the corresponding chimeric antibody (Yokota et al.,Cancer Res. 52, 3402-08 (1992)). Fusion of an scFv to another molecule,such as a toxin, takes advantage of the specific antigen-bindingactivity and the small size of an scFv to deliver the toxin to a targettissue. (Chaudary et al., Nature 339:394 (1989); Batra et al., Mol.Cell. Biol. 11:2200 (1991).)

Despite the advantages that scFv molecules bring to serotherapy, severaldrawbacks to this therapeutic approach exist. While rapid clearance ofscFv may reduce toxic effects in normal cells, such rapid clearance mayprevent delivery of a minimum effective dose to the target tissue.Manufacturing adequate amounts of scFv for administration to patientshas been challenging due to difficulties in expression and isolation ofscFv that adversely affect the yield. During expression, scFv moleculeslack stability and often aggregate due to pairing of variable regionsfrom different molecules. Furthermore, production levels of scFvmolecules in mammalian expression systems are low, limiting thepotential for efficient manufacturing of scFv molecules for therapy(Davis et al, J. Biol. Chem. 265:10410-18 (1990); Traunecker et al.,EMBO J. 10: 3655-59 (1991)). Strategies for improving production havebeen explored, including addition of glycosylation sites to the variableregions (e.g., U.S. Pat. No. 5,888,773; Jost et al, J. Biol. Chem. 269:26267-73 (1994)).

An additional disadvantage to using scFv for therapy is the lack ofeffector function. An scFv that lacks the cytolytic functions, ADCC andcomplement dependent-cytotoxicity (CDC), which are typically associatedwith immunoglobulin constant regions, may be ineffective for treatingdisease. Even though development of scFv technology began over 12 yearsago, currently no scFv products are approved for therapy. Conjugation orfusion of toxins to scFV has thus been an alternative strategy toprovide a potent, antigen-specific molecule, but dosing with suchconjugates or chimeras is often limited by excessive and/or non-specifictoxicity having its origin in the toxin moiety of such preparations.Toxic effects may include supraphysiological elevation of liver enzymesand vascular leak syndrome, and other undesired effects. In addition,immunotoxins are themselves highly immunogenic after being administeredto a host, and host antibodies generated against the immunotoxin limitits potential usefulness in repeated therapeutic treatments of anindividual.

The benefits of immunoglobulin constant region-associated effectorfunctions in the treatment of disease has prompted development of fusionproteins in which immunoglobulin constant region polypeptide sequencesare present and nonimmunoglobulin sequences are substituted for theantibody variable region. For example, CD4, the T cell surface proteinrecognized by HIV, was recombinantly fused to an immunoglobulin Fceffector domain. (See Sensel et al., Chem. Immunol. 65:129-158 (1997).)The biological activity of such a molecule will depend in part on theclass or subclass of the constant region chosen. An IL-2-IgG1 fusionprotein effected complement-mediated lysis of IL-2 receptor-bearingcells. (See id.) Use of immunoglobulin constant regions to constructthese and other fusion proteins may also confer improved pharmacokineticproperties.

Diseases and disorders thought to be amenable to some type ofimmunoglobulin therapy include cancer and immune system disorders.Cancer includes a broad range of diseases, affecting approximately onein four individuals worldwide. Rapid and unregulated proliferation ofmalignant cells is a hallmark of many types of cancer, includinghematological malignancies. Patients with a hematologic malignantcondition have benefited most from advances in cancer therapy in thepast two decades (Multani et al., J. Clin. Oncology 16: 3691-3710,1998). Although remission rates have increased, most patients stillrelapse and succumb to their disease. Barriers to cure with cytotoxicdrugs include tumor cell resistance and the high toxicity ofchemotherapy, which prevents optimal dosing in many patients. Newtreatments based on targeting with molecules that specifically bind to amalignant cell, including monoclonal antibodies (mAbs), can improveeffectiveness without increasing toxicity.

Since monoclonal antibodies (mAb) were first described in 1975 (Kohleret al., Nature 256:495-97 (1975)), many patients have been treated withmAbs that specifically bind to tumor antigens, or antigens expressed ontumor cells. These studies have yielded important lessons regarding theselection of tumor cell surface antigens that are tumor antigenssuitable for use as immunotherapy targets. First, it is highlypreferable that such a target antigen is not expressed by normal tissuesthe preservation of which is important to host survival. Fortunately, inthe case of hematologic malignancy, malignant cells express manyantigens that are not expressed on the surfaces of stem cells or otheressential cells. Treatment of a hematologic malignant condition using atherapeutic regimen that depletes both normal and malignant cells ofhematological origin has been acceptable where regeneration of normalcells from progenitors can occur after therapy has ended. Second, thetarget antigen should be expressed on all or virtually all clonogenicpopulations of tumor cells, and expression should persist despite theselective pressure from immunoglobulin therapy. Thus, a strategy thatemploys selection of a cell surface idiotype (e.g., a particularidiotope) as a target for therapy of B cell malignancy has been limitedby the outgrowth of tumor cell variants with altered surface idiotypeexpression, even where the antigen exhibits a high degree of tumorselectivity (Meeker et al., N. Engl. J. Med. 312:1658-65 (1985)). Third,the selected antigen must traffic properly after an immunoglobulin bindsto it. Shedding or internalization of a cell surface target antigenafter an immunoglobulin binds to the antigen may allow tumor cells toescape destruction, thus limiting the effectiveness of serotherapy.Fourth, binding of an immunoglobulin to cell surface target antigensthat transmit or transduce cellular activation signals may result inimproved functional responses to immunotherapy in tumor cells, and canlead to growth arrest and/or apoptosis. While all of these propertiesare important, the triggering of apoptosis after an immunoglobulin bindsto the target antigen may be a critical factor in achieving successfulserotherapy. Antigens that have been tested as targets for serotherapyof B and T cell malignancies include Ig idiotype (Brown et al., Blood73:651-61 (1989)), CD19 (Hekman et al., Cancer Immunol. Immunother.32:364-72 (1991); Vlasveld et al., Cancer Immunol. Immunother. 40: 37-47(1995)), CD20 (Press et al., Blood 69: 584-91 (1987); Maloney et al., J.Clin. Oncol. 15:3266-74, (1997)) CD21 (Scheinberg et. al., J. Clin.Oncol. 8:792-803, (1990)), CD5 (Dillman et. al., J. Biol. Respn. Mod.5:394-410 (1986)), and CD52 (CAMPATH) (Pawson et al., J. Clin. Oncol.15:2667-72, (1997)). Of these, the most success has been obtained usingCD20 as a target for therapy of B cell lymphomas. Each of the othertargets has been limited by the biological properties of the antigen.For example, surface idiotype can be altered through somatic mutation,allowing tumor cell escape. As other examples, CD5, CD21, and CD19 arerapidly internalized after mAb binding, allowing tumor cells to escapedestruction unless mAbs are conjugated with toxin molecules. CD22 isexpressed on only a subset of B cell lymphomas, thereby limiting itsusefulness, while CD52 is expressed on both T cells and B cells and maytherefore generate counterproductive immunosuppression by effectingselective T cell depletion.

CD20 fulfills the basic criteria described above for selection of anappropriate target antigen for therapy of a B cell malignant condition.Treatment of patients with low grade or follicular B cell lymphoma usingchimeric CD20 mAb induces partial or complete responses in many patients(McLaughlin et al, Blood 88:90a (abstract, suppl. 1) (1996); Maloney etal, Blood 90: 2188-95 (1997)). However, tumor relapse commonly occurswithin six months to one year. Therefore, further improvements inserotherapy are needed to induce more durable responses in low grade Bcell lymphoma, and to allow effective treatment of high grade lymphomaand other B cell diseases.

One approach to improving CD20 serotherapy has been to targetradioisotopes to B cell lymphomas using mAbs specific for CD20. Whilethe effectiveness of therapy is increased, associated toxicity from thelong in vivo half-life of the radioactive antibody increases also,sometimes requiring that the patient undergo stem cell rescue (Press etal., N. Eng. J. Med. 329: 1219-1224, 1993; Kaminski et al., N. Eng. J.Med. 329:459-65 (1993)). MAbs to CD20 have been cleaved with proteasesto yield F(ab′)₂ or Fab fragments prior to attachment of theradioisotope. This improves penetration of the radioisotope conjugateinto the tumor, and shortens the in vivo half-life, thus reducing thetoxicity to normal tissues. However, the advantages of effectorfunctions, including complement fixation and/or ADCC that wouldotherwise be provided by the Fc region of the CD20 mAb, are lost sincethe Fab preparations lack immunoglobulin Fc domains. Therefore, forimproved delivery of radioisotopes, a strategy is needed to make a CD20mAb derivative that retains Fc-dependent effector functions but which issmaller in size, thereby increasing tumor penetration and shortening mAbhalf-life.

CD20 was the first human B cell lineage-specific surface moleculeidentified by a monoclonal antibody, but the function of CD20 in B cellbiology is still incompletely understood. CD20 is a non-glycosylated,hydrophobic 35 kDa B cell transmembrane phosphoprotein that has bothamino and carboxy ends situated in the cytoplasm (Einfeld et al, EMBO J.7:711-17 (1988)). Natural ligands for CD20 have not been identified.CD20 is expressed by all normal mature B cells, but is not expressed byprecursor B cells.

CD20 mAbs deliver signals to normal B cells that affect viability andgrowth (Clark et al., Proc. Natl. Acad. Sci. USA 83:4494-98 (1986)), andextensive cross-linking of CD20 can induce apoptosis in B lymphoma celllines (Shan et al., Blood 91:1644-52 (1998)). Cross-linking of CD20 onthe cell surface increases the magnitude and enhances the kinetics ofsignal transduction, for example, as detected by measuring tyrosinephosphorylation of cellular substrates (Deans et al., J. Immunol.146:846-53 (1993)). Significantly, apoptosis in Ramos B lymphoma cellscan also be induced by FcR cross-linking CD20 mAbs bound to the Ramoscell surfaces, by the addition of Fc-receptor positive cells (Shan etal., Blood 91: 1644-52 (1998)). Therefore, in addition to cellulardepletion by complement and ADCC mechanisms, Fc-receptor binding by CD20mAbs in vivo can promote apoptosis of malignant B cells by CD20cross-linking This theory is consistent with experiments showing thateffectiveness of CD20 therapy of human lymphoma in a SCID mouse modelwas dependent upon Fc-receptor binding by the CD20 mAb (Funakoshi etal., J. Immunotherapy 19:93-101 (1996)).

The CD20 polypeptide contains four transmembrane domains (Einfeld etal., EMBO J. 7: 711-17, (1988); Stamenkovic et al., J. Exp. Med.167:1975-80 (1988); Tedder et. al., J. Immunol. 141:4388-4394 (1988)).The multiple membrane spanning domains prevent CD20 internalizationafter antibody binding. This property of CD20 was recognized as animportant feature for effective therapy of B cell malignancies when amurine CD20 mAb, 1F5, was injected into patients with B cell lymphoma,resulting in significant depletion of malignant cells and partialclinical responses (Press et al., Blood 69: 584-91 (1987)).

Because normal mature B cells also express CD20, normal B cells aredepleted during CD20 antibody therapy (Reff, M. E. et al, Blood 83:435-445, 1994). However, after treatment is completed, normal B cellsare regenerated from CD20 negative B cell precursors; therefore,patients treated with anti-CD20 therapy do not experience significantimmunosuppression. Depletion of normal B cells may also be beneficial indiseases that involve inappropriate production of autoantibodies orother diseases where B cells may play a role. A chimeric mAb specificfor CD20, consisting of heavy and light chain variable regions of mouseorigin fused to human IgG1 heavy chain and human kappa light chainconstant regions, retained binding to CD20 and the ability to mediateADCC and to fix complement (Liu et al., J. Immunol. 139:3521-26 (1987);Robinson et al., U.S. Pat. No. 5,500,362). This work led to developmentof a chimeric CD20 mAb, Rituximab™, currently approved by the U.S. Foodand Drug Administration for approval for therapy of B cell lymphomas.While clinical responses are frequently observed after treatment withRituximab™, patients often relapse after about 6-12 months.

High doses of Rituximab™ are required for intravenous injection becausethe molecule is large, approximately 150 kDa, and diffusion is limitedinto the lymphoid tissues where many tumor cells reside. The mechanismof anti-tumor activity of Rituximab™ is thought to be a combination ofseveral activities, including ADCC, complement fixation, and triggeringof signals that promote apoptosis in malignant B cells. The large sizeof Rituximab™ prevents optimal diffusion of the molecule into lymphoidtissues that contain malignant B cells, thereby limiting theseanti-tumor activities. As discussed above, cleavage of CD20 mAbs withproteases into Fab or F(ab′)₂ fragments makes them smaller and allowsbetter penetration into lymphoid tissues, but the effector functionsimportant for anti-tumor activity are lost. While CD20 mAb fragments maybe more effective than intact antibody for delivery of radioisotopes, itwould be desirable to construct a CD20 mAb derivative that retains theeffector functions of the Fc portion, but that has a smaller molecularsize, facilitating better tumor penetration and resulting in a shorterhalf-life.

CD20 is expressed by many malignant cells of B cell origin, including Bcell lymphoma and chronic lymphocytic leukemia (CLL). CD20 is notexpressed by malignancies of pre-B cells, such as acute lymphoblasticleukemia. CD20 is therefore a good target for therapy of B celllymphoma, CLL, and other diseases in which B cells are involved in thepathogenesis and/or progression of disease. Other B cell disordersinclude autoimmune diseases in which autoantibodies are produced duringor after the differentiation of B cells into plasma cells. Examples of Bcell disorders include autoimmune thyroid disease, including Graves'disease and Hashimoto's thyroiditis, rheumatoid arthritis, systemiclupus erythematosus (SLE), Sjogrens syndrome, immune thrombocytopenicpurpura (ITP), multiple sclerosis (MS), myasthenia gravis (MG),psoriasis, scleroderma, and inflammatory bowel disease, includingCrohn's disease and ulcerative colitis.

In view of the foregoing, there is clearly a need for improvedcompositions and methods to treat malignant conditions in general, andin particular B cell disorders. As described in greater detail herein,the compositions and methods of the present invention overcome thelimitations of the prior art by providing a bindingdomain-immunoglobulin fusion protein that specifically binds to anantigen and that is capable of mediating ADCC or complement fixation.Furthermore, the compositions and methods offer other relatedadvantages.

BRIEF SUMMARY

It is an aspect of the present invention to provide a bindingdomain-immunoglobulin fusion protein, comprising (a) a binding domainpolypeptide that is fused to an immunoglobulin hinge region polypeptide,wherein said hinge region polypeptide is selected from the groupconsisting of (i) a wild-type human IgG1 immunoglobulin hinge regionpolypeptide, (ii) a mutated human IgG1 immunoglobulin hinge regionpolypeptide that is derived from a wild-type immunoglobulin hinge regionpolypeptide having three or more cysteine residues, wherein said mutatedhuman IgG1 immunoglobulin hinge region polypeptide contains two cysteineresidues and wherein a first cysteine of the wild-type hinge region isnot mutated, (iii) a mutated human IgG1 immunoglobulin hinge regionpolypeptide that is derived from a wild-type immunoglobulin hinge regionpolypeptide having three or more cysteine residues, wherein said mutatedhuman IgG1 immunoglobulin hinge region polypeptide contains no more thanone cysteine residue, and (iv) a mutated human IgG1 immunoglobulin hingeregion polypeptide that is derived from a wild-type immunoglobulin hingeregion polypeptide having three or more cysteine residues, wherein saidmutated human IgG1 immunoglobulin hinge region polypeptide contains nocysteine residues; (b) an immunoglobulin heavy chain CH2 constant regionpolypeptide that is fused to the hinge region polypeptide; and (c) animmunoglobulin heavy chain CH3 constant region polypeptide that is fusedto the CH2 constant region polypeptide, wherein (1) the bindingdomain-immunoglobulin fusion protein is capable of at least oneimmunological activity selected from the group consisting of antibodydependent cell-mediated cytotoxicity and complement fixation, and (2)the binding domain polypeptide is capable of specifically binding to anantigen.

In certain embodiments the immunoglobulin hinge region polypeptide is amutated hinge region polypeptide and exhibits a reduced ability todimerize, relative to a wild-type human immunoglobulin G hinge regionpolypeptide. In certain embodiments the binding domain polypeptidecomprises at least one immunoglobulin variable region polypeptide thatis selected an immunoglobulin light chain variable region polypeptide oran immunoglobulin heavy chain variable region polypeptide. In certainfurther embodiments the binding domain-immunoglobulin fusion proteincomprises an immunoglobulin heavy chain variable region polypeptide,wherein the heavy chain variable region polypeptide is a humanimmunoglobulin heavy chain variable region polypeptide comprising amutation at an amino acid at a location corresponding to amino acidposition 11 in the VH domain, or amino acid position 155 in SEQ ID NO:11, position 158 in SEQ ID NO: 12, position 154 in SEQ ID NO:13, orposition 159 in SEQ ID NO 14, or amino acid position 11 in the VH domainpolypeptides listed in SEQ ID NOS: 341, 354, 465, 471, 477,548. Incertain embodiments the immunoglobulin variable region polypeptide isderived from a human immunoglobulin, and in certain other embodimentsthe immunoglobulin variable region polypeptide comprises a humanizedimmunoglobulin polypeptide sequence. In certain embodiments theimmunoglobulin variable region polypeptide is derived from a murineimmunoglobulin.

According to certain embodiments of the present invention, the bindingdomain polypeptide comprises (a) at least one immunoglobulin light chainvariable region polypeptide; (b) at least one immunoglobulin heavy chainvariable region polypeptide; and (c) at least one linker polypeptidethat is fused to the polypeptide of (a) and to the polypeptide of (b).In certain further embodiments the immunoglobulin light chain variableregion and heavy chain variable region polypeptides are derived fromhuman immunoglobulins, and in certain other further embodiments thelinker polypeptide comprises at least one polypeptide having as an aminoacid sequence Gly-Gly-Gly-Gly-Ser [SEQ ID NO: 39]. In other embodimentsthe linker polypeptide comprises at least three repeats of a polypeptidehaving as an amino acid sequence Gly-Gly-Gly-Gly-Ser [SEQ ID NO: 39]. Inother embodiments the linker comprises a glycosylation site, which incertain further embodiments is an asparagine-linked glycosylation site,an O-linked glycosylation site, a C-mannosylation site, a glypiationsite or a phosphoglycation site. In another embodiment at least one ofthe immunoglobulin heavy chain CH2 constant region polypeptide and theimmunoglobulin heavy chain CH3 constant region polypeptide is derivedfrom a human immunoglobulin heavy chain. In another embodiment theimmunoglobulin heavy chain constant region CH2 and CH3 polypeptides areof an isotype that is human IgG or human IgA. In certain otherembodiments the antigen is CD19, CD20, CD22, CD37, CD40, L6, CD2, CD28,CD30, CD40, LD50 (ICAM3), CD54 (ICAM1), CD80, CD86, B7-H1, CD134 (OX40),CD137 (41BB), CD152 (CTLA-4), CD153 (CD30 ligand), CD154 (CD40 ligand),ICOS, CD19, CD3, CD4, CD25, CD8, CD11b, CD14, CD25, CD56 or CD69. Inanother embodiment the binding domain polypeptide comprises a CD154extracellular domain. In still another embodiment the binding domainpolypeptide comprises a CD154 extracellular domain and at least oneimmunoglobulin variable region polypeptide. In another embodiment thebinding domain polypeptide comprises a CTLA-4 extracellular domain, andin further embodiments at least one of the immunoglobulin heavy chainconstant region polypeptides selected from a CH2 constant regionpolypeptide and a CH3 constant region polypeptide is a human IgG1constant region polypeptide. In another further embodiment at least oneof the immunoglobulin heavy chain constant region polypeptides selectedfrom a CH2 constant region polypeptide and a CH3 constant regionpolypeptide is a human IgA constant region polypeptide.

Turning to another embodiment, the present invention provides a bindingdomain-immunoglobulin fusion protein, comprising (a) a binding domainpolypeptide that is fused to an immunoglobulin hinge region polypeptide;(b) an immunoglobulin heavy chain CH2 constant region polypeptide thatis fused to the hinge region polypeptide; and (c) an immunoglobulinheavy chain CH3 constant region polypeptide that is fused to the CH2constant region polypeptide, wherein (1) the binding domain polypeptidecomprises a CTLA-4 extracellular domain that is capable of specificallybinding to at least one CTLA-4 ligand selected from the group consistingof CD80 and CD86, (2) the immunoglobulin hinge region polypeptidecomprises a polypeptide that is selected from the group consisting of ahuman IgA hinge region polypeptide and a human IgG1 hinge regionpolypeptide, (3) the immunoglobulin heavy chain CH2 constant regionpolypeptide comprises a polypeptide that is selected from the groupconsisting of a human IgA heavy chain CH2 constant region polypeptideand a human IgG1 heavy chain CH2 constant region polypeptide, (4) theimmunoglobulin heavy chain CH3 constant region polypeptide comprises apolypeptide that is selected from the group consisting of a human IgAheavy chain CH3 constant region polypeptide and a human IgG1 heavy chainCH3 constant region polypeptide, and (5) the bindingdomain-immunoglobulin fusion protein is capable of at least oneimmunological activity selected from the group consisting of antibodydependent cell-mediated cytotoxicity and complement fixation.

In another embodiment the present invention provides a bindingdomain-immunoglobulin fusion protein, comprising (a) a binding domainpolypeptide that is fused to an immunoglobulin hinge region polypeptide,wherein said hinge region polypeptide comprises a human IgE hinge regionpolypeptide; (b) an immunoglobulin heavy chain CH2 constant regionpolypeptide that is fused to the hinge region polypeptide, wherein saidCH2 constant region polypeptide comprises a human IgE CH2 constantregion polypeptide; and (c) an immunoglobulin heavy chain CH3 constantregion polypeptide that is fused to the CH2 constant region polypeptide,wherein said CH3 constant region polypeptide comprises a human IgE CH3constant region polypeptide wherein (1) the bindingdomain-immunoglobulin fusion protein is capable of at least oneimmunological activity selected from antibody dependent cell-mediatedcytotoxicity and induction of an allergic response mechanism, and (2)the binding domain polypeptide is capable of specifically binding to anantigen. In a further embodiment the binding domain-immunoglobulinfusion protein comprises a human IgE CH4 constant region polypeptide. Inanother further embodiment the antigen is a tumor antigen.

In certain other embodiments the present invention provides a bindingdomain-immunoglobulin fusion protein, comprising (a) a binding domainpolypeptide that is fused to an immunoglobulin hinge region polypeptide,wherein the binding domain polypeptide is capable of specificallybinding to at least one antigen that is present on an immune effectorcell and wherein the hinge region polypeptide comprises a polypeptideselected from the group consisting of a human IgA hinge regionpolypeptide, a human IgG hinge region polypeptide, and a human IgE hingeregion polypeptide; (b) an immunoglobulin heavy chain CH2 constantregion polypeptide that is fused to the hinge region polypeptide,wherein said CH2 constant region polypeptide comprises a polypeptideselected from the group consisting of a human IgA CH2 constant regionpolypeptide, a human IgG CH2 constant region polypeptide, and a humanIgE CH2 constant region polypeptide; (c) an immunoglobulin heavy chainCH3 constant region polypeptide that is fused to the CH2 constant regionpolypeptide, wherein said CH3 constant region polypeptide comprises apolypeptide selected from the group consisting of a human IgA CH3constant region polypeptide, a human IgG CH3 constant regionpolypeptide, and a human IgE CH3 constant region polypeptide; and (d) aplasma membrane anchor domain polypeptide. In a further embodiment themembrane anchor domain polypeptide comprises a transmembrane domainpolypeptide. In another further embodiment the membrane anchor domainpolypeptide comprises a transmembrane domain polypeptide and acytoplasmic tail polypeptide. In a still further embodiment thecytoplasmic tail polypeptide comprises an apoptosis signalingpolypeptide sequence, which in a still further embodiment is derivedfrom a receptor death domain polypeptide. In a further embodiment thedeath domain polypeptide comprises a polypeptide selected from an ITIMdomain, an ITAM domain, FADD, TRADD, RAIDD, CD95 (FAS/Apo-1), TNFR1 orDRS. In another embodiment the apoptosis signaling polypeptide sequencecomprises a polypeptide sequence derived from a caspase polypeptide thatis caspase-3 or caspase-8. In another embodiment the plasma membraneanchor domain polypeptide comprises aglycosyl-phosphatidylinositol-linkage polypeptide sequence. In anotherembodiment the antigen that is present on an immune effector cell isCD2, CD28, CD30, CD40, LD50 (ICAM3), CD54 (ICAM1), CD80, CD86, B7-H1,CD134 (OX40), CD137 (41BB), CD152 (CTLA-4), CD153 (CD30 ligand), CD154(CD40 ligand), ICOS, CD19, CD20, CD22, CD37, L6, CD3, CD4, CD25, CD8,CD11b, CD14, CD25, CD56 or CD69. In another embodiment the human IgG ishuman IgG1.

The invention provides, in another embodiment, a bindingdomain-immunoglobulin fusion protein, comprising (a) a binding domainpolypeptide that is fused to an immunoglobulin hinge region polypeptide,wherein the binding domain polypeptide is capable of specificallybinding to at least one antigen that is present on a cancer cell surfaceand wherein the hinge region polypeptide comprises a polypeptideselected from the group consisting of a human IgA hinge regionpolypeptide, a human IgG hinge region polypeptide, and a human IgE hingeregion polypeptide; (b) an immunoglobulin heavy chain CH2 constantregion polypeptide that is fused to the hinge region polypeptide,wherein the CH2 constant region polypeptide comprises a polypeptide thatis a human IgA CH2 constant region polypeptide, a human IgG CH2 constantregion polypeptide, or a human IgE CH2 constant region polypeptide; (c)an immunoglobulin heavy chain CH3 constant region polypeptide that isfused to the CH2 constant region polypeptide, wherein the CH3 constantregion polypeptide comprises a polypeptide that is a human IgA CH3constant region polypeptide, a human IgG CH3 constant regionpolypeptide, or a human IgE CH3 constant region polypeptide; and (d) aplasma membrane anchor domain polypeptide. In a further embodiment themembrane anchor domain polypeptide comprises a transmembrane domainpolypeptide. In another embodiment the membrane anchor domainpolypeptide comprises a transmembrane domain polypeptide and acytoplasmic tail polypeptide. In another embodiment the membrane anchordomain polypeptide comprises a glycosyl-phosphatidylinositol-linkagepolypeptide sequence. In another embodiment the human IgG is human IgG1.

In another embodiment the present invention provides a bindingdomain-immunoglobulin fusion protein, comprising (a) a binding domainpolypeptide that is fused to an immunoglobulin hinge region polypeptide,wherein said hinge region polypeptide comprises a wild-type human IgAhinge region polypeptide; (b) an immunoglobulin heavy chain CH2 constantregion polypeptide that is fused to the hinge region polypeptide,wherein said CH2 constant region polypeptide comprises a human IgA CH2constant region polypeptide; and (c) an immunoglobulin heavy chain CH3constant region polypeptide that is fused to the CH2 constant regionpolypeptide, wherein the CH3 constant region polypeptide comprises apolypeptide that is (i) a wild-type human IgA CH3 constant regionpolypeptide or (ii) a mutated human IgA CH3 constant region polypeptidethat is incapable of associating with a J chain, wherein (1) the bindingdomain-immunoglobulin fusion protein is capable of at least oneimmunological activity selected from the group consisting of antibodydependent cell-mediated cytotoxicity and complement fixation, and (2)the binding domain polypeptide is capable of specifically binding to anantigen. In certain further embodiments the mutated human IgA CH3constant region polypeptide that is incapable of associating with a Jchain is (i) a polypeptide comprising an amino acid sequence as setforth in SEQ ID NOS: 296, 511AA or 295 and 510 (DNA) or (ii) apolypeptide comprising an amino acid sequence as set forth in SEQ ID NO:303, 520 for amino acid sequence, and (302, 519 for DNA).

In certain other embodiments the present invention provides a bindingdomain-immunoglobulin fusion protein, comprising (a) a binding domainpolypeptide that is fused to an immunoglobulin hinge region polypeptide;(b) an immunoglobulin heavy chain CH2 constant region polypeptide thatis fused to the hinge region polypeptide, wherein the CH2 constantregion polypeptide comprises a llama CH2 constant region polypeptidethat is a llama IgG1 CH2 constant region polypeptide, a llama IgG2 CH2constant region polypeptide or a llama IgG3 CH2 constant regionpolypeptide; and (c) an immunoglobulin heavy chain CH3 constant regionpolypeptide that is fused to the CH2 constant region polypeptide,wherein said CH3 constant region polypeptide comprises a llama CH3constant region polypeptide that is selected from the group consistingof a llama IgG1 CH3 constant region polypeptide, a llama IgG2 CH3constant region polypeptide and a llama IgG3 CH3 constant regionpolypeptide wherein (1) the binding domain-immunoglobulin fusion proteinis capable of at least one immunological activity selected from thegroup consisting of antibody dependent cell-mediated cytotoxicity andinduction fixation of complement, and (2) the binding domain polypeptideis capable of specifically binding to an antigen. In a furtherembodiment the immunoglobulin hinge region polypeptide, the llama CH2constant region polypeptide and the llama CH3 constant regionpolypeptide comprise sequences derived from a llama IgG1 polypeptide andthe fusion protein does not include a llama IgG1 CH1 domain. In certainembodiments the invention provides any of the above described bindingdomain-immunoglobulin fusion proteins wherein the hinge regionpolypeptide is mutated to contain a glycosylation site, which in certainfurther embodiments is an asparagine-linked glycosylation site, anO-linked glycosylation site, a C-mannosylation site, a glypiation siteor a phosphoglycation site. In certain embodiments the inventionprovides any of the above described binding domain-immunoglobulin fusionproteins wherein the binding domain polypeptide comprises two or morebinding domain polypeptide sequences wherein each of the binding domainpolypeptide sequences is capable of specifically binding to an antigen.

The present invention also provides, in certain embodiments, a bindingdomain-immunoglobulin fusion protein, comprising (a) a binding domainpolypeptide that is fused to an immunoglobulin hinge region polypeptide,wherein the hinge region polypeptide comprises an alternative hingeregion polypeptide sequence; (b) an immunoglobulin heavy chain CH2constant region polypeptide that is fused to the hinge regionpolypeptide; and (c) an immunoglobulin heavy chain CH3 constant regionpolypeptide that is fused to the CH2 constant region polypeptide,wherein: (1) the binding domain-immunoglobulin fusion protein is capableof at least one immunological activity selected from the groupconsisting of antibody dependent cell-mediated cytotoxicity andcomplement fixation, and (2) the binding domain polypeptide is capableof specifically binding to an antigen.

Turning to another embodiment there is provided a bindingdomain-immunoglobulin fusion protein, comprising (a) a binding domainpolypeptide that is fused to an immunoglobulin hinge region polypeptide,wherein the binding domain polypeptide is capable of specificallybinding to at least one antigen that is present on a cancer cell surfaceand wherein the hinge region polypeptide comprises an alternative hingeregion polypeptide sequence; (b) an immunoglobulin heavy chain CH2constant region polypeptide that is fused to the hinge regionpolypeptide, wherein said CH2 constant region polypeptide comprises apolypeptide selected from the group consisting of a human IgA CH2constant region polypeptide, a human IgG CH2 constant regionpolypeptide, and a human IgE CH2 constant region polypeptide; (c) animmunoglobulin heavy chain CH3 constant region polypeptide that is fusedto the CH2 constant region polypeptide, wherein the CH3 constant regionpolypeptide comprises a polypeptide that is a human IgA CH3 constantregion polypeptide, a human IgG CH3 constant region polypeptide, or ahuman IgE CH3 constant region polypeptide; and (d) a plasma membraneanchor domain polypeptide. In certain further embodiments thealternative hinge region polypeptide sequence comprises a polypeptidesequence of at least ten continuous amino acids that are present in asequence selected from SEQ ID NOS: 215, 216, 217, 218, 223,224, 6, 15,16. 35, 36, 37, 41,207, 208, 223, 275, 276, 277, 296, 300, 350, 390,391, 392, 396, 397, 398, 488, 582, 584, 586.

In certain embodiments the present invention provides an isolatedpolynucleotide encoding any one of the above described bindingdomain-immunoglobulin fusion proteins, and in other embodiments theinvention provides a recombinant expression construct comprising anysuch polynucleotide that is operably linked to a promoter. In otherembodiments there is provided a host cell transformed or transfectedwith any such recombinant expression construct. In a related embodimentthere is provided a method of producing a binding domain-immunoglobulinfusion protein, comprising the steps of (a) culturing a host cell asjust described under conditions that permit expression of the bindingdomain-immunoglobulin fusion protein; and (b) isolating the bindingdomain-immunoglobulin fusion protein from the host cell culture. Inanother embodiment there is provided a pharmaceutical compositioncomprising any one of the above described binding domain-immunoglobulinfusion proteins in combination with a physiologically acceptablecarrier. In another embodiment the invention provides a pharmaceuticalcomposition comprising an isolated polynucleotide encoding any one ofthe above described binding domain-immunoglobulin fusion proteins, incombination with a physiologically acceptable carrier. In anotherembodiment the invention provides a method of treating a subject havingor suspected of having a malignant condition or a B-cell disorder,comprising administering to a patient a therapeutically effective amountof any of the pharmaceutical compositions just described. In certainfurther embodiments the malignant condition or B-cell disorder is aB-cell lymphoma or a disease characterized by autoantibody production,and in certain other further embodiments the malignant condition orB-cell disorder is rheumatoid arthritis, myasthenia gravis, Grave'sdisease, type I diabetes mellitus, multiple sclerosis or an autoimmunedisease. In certain other embodiments the malignant condition ismelanoma, carcinoma or sarcoma.

It is another aspect of the present invention to provide a bindingdomain-immunoglobulin fusion protein, comprising (a) a binding domainpolypeptide that is fused to an immunoglobulin hinge region polypeptide,wherein said hinge region polypeptide is selected from the groupconsisting of (i) a mutated hinge region polypeptide that contains nocysteine residues and that is derived from a wild-type immunoglobulinhinge region polypeptide having one or more cysteine residues, (ii) amutated hinge region polypeptide that contains one cysteine residue andthat is derived from a wild-type immunoglobulin hinge region polypeptidehaving two or more cysteine residues, (iii) a wild-type human IgA hingeregion polypeptide, (iv) a mutated human IgA hinge region polypeptidethat contains no cysteine residues and that is derived from a wild-typehuman IgA region polypeptide, and (v) a mutated human IgA hinge regionpolypeptide that contains one cysteine residue and that is derived froma wild-type human IgA region polypeptide; (b) an immunoglobulin heavychain CH2 constant region polypeptide that is fused to the hinge regionpolypeptide; and (c) an immunoglobulin heavy chain CH3 constant regionpolypeptide that is fused to the CH2 constant region polypeptide,wherein: (1) the binding domain-immunoglobulin fusion protein is capableof at least one immunological activity selected from the groupconsisting of antibody dependent cell-mediated cytotoxicity andcomplement fixation, and (2) the binding domain polypeptide is capableof specifically binding to an antigen. In one embodiment theimmunoglobulin hinge region polypeptide is a mutated hinge regionpolypeptide and exhibits a reduced ability to dimerize, relative to awild-type human immunoglobulin G hinge region polypeptide. In anotherembodiment the binding domain polypeptide comprises at least oneimmunoglobulin variable region polypeptide that is an immunoglobulinlight chain variable region polypeptide or an immunoglobulin heavy chainvariable region polypeptide. In a further embodiment the immunoglobulinvariable region polypeptide is derived from a human immunoglobulin.

In another embodiment the binding domain Fv-immunoglobulin fusionprotein binding domain polypeptide comprises (a) at least oneimmunoglobulin light chain variable region polypeptide; (b) at least oneimmunoglobulin heavy chain variable region polypeptide; and (c) at leastone linker peptide that is fused to the polypeptide of (a) and to thepolypeptide of (b). In a further embodiment the immunoglobulin lightchain variable region and heavy chain variable region polypeptides arederived from human immunoglobulins.

In another embodiment at least one of the immunoglobulin heavy chain CH2constant region polypeptide and the immunoglobulin heavy chain CH3constant region polypeptide is derived from a human immunoglobulin heavychain. In another embodiment the immunoglobulin heavy chain constantregion CH2 and CH3 polypeptides are of an isotype selected from humanIgG and human IgA. In another embodiment the antigen is selected fromthe group consisting of CD19, CD20, CD37, CD40 and L6. In certainfurther embodiments of the above described fusion protein, the linkerpolypeptide comprises at least one polypeptide having as an amino acidsequence Gly-Gly-Gly-Gly-Ser [SEQ ID NO: 39], and in certain otherembodiments the linker polypeptide comprises at least three repeats of apolypeptide having as an amino acid sequence Gly-Gly-Gly-Gly-Ser [SEQ IDNO: 39]. In certain embodiments the immunoglobulin hinge regionpolypeptide comprises a human IgA hinge region polypeptide. In certainembodiments the binding domain polypeptide comprises a CD154extracellular domain. In certain embodiments the binding domainpolypeptide comprises a CD154 extracellular domain and at least oneimmunoglobulin variable region polypeptide.

In other embodiments the invention provides an isolated polynucleotideencoding any of the above described binding domain-immunoglobulin fusionproteins, and in related embodiments the invention provides arecombinant expression construct comprising such a polynucleotide, andin certain further embodiments the invention provides a host celltransformed or transfected with such a recombinant expression construct.In another embodiment the invention provides a method of producing abinding domain-immunoglobulin fusion protein, comprising the steps of(a) culturing the host cell as just described, under conditions thatpermit expression of the binding domain-immunoglobulin fusion protein;and (b) isolating the binding domain-immunoglobulin fusion protein fromthe host cell culture.

The present invention also provides in certain embodiments apharmaceutical composition comprising a binding domain-immunoglobulinfusion protein as described above, in combination with a physiologicallyacceptable carrier. In another embodiment there is provided a method oftreating a subject having or suspected of having a malignant conditionor a B-cell disorder, comprising administering to a patient atherapeutically effective amount of an above described bindingdomain-immunoglobulin fusion protein. In certain further embodiments themalignant condition or B-cell disorder is a B-cell lymphoma or a diseasecharacterized by autoantibody production, and in certain other furtherembodiments the malignant condition or B-cell disorder is rheumatoidarthritis, myasthenia gravis, Grave's disease, type I diabetes mellitus,multiple sclerosis or an autoimmune disease.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All references disclosed herein are hereby incorporated byreference in their entireties as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows DNA and deduced amino acid sequences (SEQ ID NO: 2) of2H7scFv-Ig, a binding domain-immunoglobulin fusion protein capable ofspecifically binding CD20.

FIG. 2 shows production levels of 2H7 scFv-Ig by transfected, stable CHOlines and generation of a standard curve by binding of purified 2H7scFv-Ig to CHO cells expressing CD20.

FIG. 3 shows SDS-PAGE analysis of multiple preparations of isolated2H7scFv-Ig protein.

FIG. 4 shows complement fixation (FIG. 4A) and mediation ofantibody-dependent cellular cytotoxicity (ADCC, FIG. 4B)) by 2H7scFv-Ig.

FIG. 5 shows the effect of simultaneous ligation of CD20 and CD40 ongrowth of normal B cells.

FIG. 6 shows the effect of simultaneous ligation of CD20 and CD40 onCD95 expression and induction of apoptosis in a B lymphoblastoid cellline.

FIG. 7 shows DNA and deduced amino acid sequences of 2H7scFv-CD154 L2(FIG. 7A-7B, SEQ ID NOS: 21 and 33) and 2H7scFv-CD154 S4 (FIG. 7C-7D,SEQ ID NOS:22 and 34) binding domain-immunoglobulin fusion proteinscapable of specifically binding CD20 and CD40.

FIG. 8 shows binding of 2H7scFv-CD154 binding domain-immunoglobulinfusion proteins to CD20+ CHO cells by flow immunocytofluorimetry.

FIG. 9 shows binding of Annexin V to B cell lines Ramos, BJAB, and T51after binding of 2H7scFv-CD154 binding domain-immunoglobulin fusionprotein to cells.

FIG. 10 shows effects on proliferation of B cell line T51 followingbinding of 2H7scFv-CD154 binding domain-immunoglobulin fusion protein.

FIG. 11 depicts schematic representations of the structures of2H7ScFv-Ig fusion proteins referred to as CytoxB or CytoxB derivatives:CytoxB-MHWTG1C (2H7 ScFv, mutant hinge, wild-type human IgG1 Fc domain),CytoxB-MHMG1C (2H7 ScFv, mutant hinge, mutated human IgG1 Fc domain) andCytoxB-IgAHWTHG1C (2H7 ScFv, human IgA-derived hinge (SEQ ID NO: 41),wild-type human IgG1 Fc domain). Arrows indicate position numbers ofamino acid residues believed to contribute to FcR binding and ADCCactivity (heavy arrows), and to complement fixation (light arrows). Noteabsence of interchain disulfide bonds.

FIG. 12 shows SDS-PAGE analysis of isolated CytoxB and 2H7scFv-CD154binding domain-immunoglobulin fusion proteins.

FIG. 13 shows antibody dependent cell-mediated cytotoxicity (ADCC)activity of CytoxB derivatives.

FIG. 14 shows complement dependent cytotoxicity (CDC) of CytoxBderivatives.

FIG. 15 shows serum half-life determinations of CytoxB-MHWTG1C inmacaque blood samples.

FIG. 16 shows effects of CytoxB-MHWTG1C on levels of circulating CD40+Bcells in macaque blood samples.

FIG. 17 shows production levels of HD37 (CD19-specific) ScFv-Ig bytransfected mammalian cell lines and generation of a standard curve bybinding of purified HD37 ScFv-Ig to cells expressing CD19.

FIG. 18 shows production levels of L6 (carcinoma antigen) ScFv-Ig bytransfected, stable CHO lines and generation of a standard curve bybinding of purified L6 ScFv-Ig to cells expressing L6 antigen.

FIG. 19 shows ADCC activity of binding domain-immunoglobulin fusionproteins 2H7 ScFv-Ig, HD37 ScFv-Ig and G28-1 (CD37-specific) ScFv-Ig.

FIG. 20 shows ADCC activity of L6 ScFv-Ig fusion proteins.

FIG. 21 shows SDS-PAGE analysis of L6 ScFv-Ig and 2H7 ScFv-Ig fusionproteins.

FIG. 22 shows SDS-PAGE analysis of G28-1 ScFv-Ig and HD37 ScFv-Ig fusionproteins.

FIG. 23 presents a sequence alignment of immunoglobulin hinge, CH2 andCH3 domains of human IgG1 (SEQ ID NO: 428) with the hinge, CH2 and CH3domains of llama IgG1 (SEQ ID NO: 430), IgG2 (SEQ ID NO: 432), and IgG3(SEQ ID NO: 434).

FIG. 24 illustrates migration of purified 2H7 scFv llama IgG fusionproteins in a 10% SDS polyacrylamide gel. Purified fusion proteins (5.mu.g per sample) were prepared in non-reducing sample buffer (lanes2-5) and in reducing sample buffer (lanes 6-9). Lane 1: molecular weightmarkers (non-reduced); lanes 2 and 6: 2H7 scFv-llama IgG1 (DNA sequenceis set forth in SEQ ID NO: 452; amino acid sequence is set forth in SEQID NO: 453); Lanes 3 and 7: 2H7 scFv-llama IgG2 (DNA sequence is setforth in SEQ ID NO: 454; amino acid sequence is set forth in SEQ ID NO:455); lanes 4 and 8: 2H7 scFv-llama IgG3 (DNA sequence is set forth inSEQ ID NO: 456; amino acid sequence is set forth in SEQ ID NO: 457); andLanes 5 and 9: Rituximab (chimeric anti-CD20 antibody (human IgG1constant region)).

FIG. 25 shows binding of 2H7 scFv-llama IgG1 (DNA sequence is set forthin SEQ ID NO: 452; amino acid sequence is set forth in SEQ ID NO: 453),2H7 scFv-llama IgG2 (DNA sequence is set forth in SEQ ID NO: 454; aminoacid sequence is set forth in SEQ ID NO: 455), and 2H7 scFv-llama IgG3(DNA sequence is set forth in SEQ ID NO: 456; amino acid sequence is setforth in SEQ ID NO: 457) to CD20+ CHO cells detected by flowimmunocytofluorimetry.

FIG. 26 depicts CDC activity of 2H7 scFv llama IgG fusion proteins, 2H7scFv-llama IgG1 (DNA sequence is set forth in SEQ ID NO: 452; amino acidsequence is set forth in SEQ ID NO: 453), 2H7 scFv-llama IgG2 (DNAsequence is set forth in SEQ ID NO: 454; amino acid sequence is setforth in SEQ ID NO: 455), and 2H7 scFv-llama IgG3 (SEQ ID NO: DNAsequence is set forth in SEQ ID NO: 454; amino acid sequence is setforth in SEQ ID NO: 455), and 2H7 scFv human IgG1 (2H7 scFv IgG WTHWTCH2CH3) (DNA sequence is set forth in SEQ ID NO: 456; amino acidsequence is set forth in SEQ ID NO: 457) against BJAB cells in thepresence of rabbit complement. Rituximab was included as a positivecontrol.

FIG. 27 shows ADCC activity of 2H7 scFv llama IgG fusion proteins, 2H7scFv-llama IgG1 (DNA sequence is set forth in SEQ ID NO: 452; amino acidsequence is set forth in SEQ ID NO: 453), 2H7 scFv-llama IgG2 (SEQ IDNO: DNA sequence is set forth in SEQ ID NO: 454; amino acid sequence isset forth in SEQ ID NO: 455), and 2H7 scFv-llama IgG3 (DNA sequence isset forth in SEQ ID NO: 456; amino acid sequence is set forth in SEQ IDNO: 457). Effector cells (human PBMC) were combined with target cells(BJAB cells) at three different ratios, 1:25, 1:50, and 1:100. Rituximabwas included as a positive control. Each data point represents threeseparate measurements.

FIG. 28 shows ADCC activity of 2H7 scFv llama IgG fusion proteins, 2H7scFv-llama IgG1 (DNA sequence is set forth in SEQ ID NO: 452; amino acidsequence is set forth in SEQ ID NO: 453), 2H7 scFv-llama IgG2 (DNAsequence is set forth in SEQ ID NO: 454; amino acid sequence is setforth in SEQ ID NO: 455), and 2H7 scFv-llama IgG3 (DNA sequence is setforth in SEQ ID NO: 456; amino acid sequence is set forth in SEQ ID NO:457). Effector cells (llama PBMC) were combined with target cells (BJABcells) at three different ratios, 1:25, 1:50, and 1:100. Rituximab wasincluded as a positive control. Each data point represents threeseparate measurements.

FIG. 29 depicts CDC activity of Reh cells (acute lymphocytic leukemia)expressing scFv-Ig fusion proteins on the cell surface. Reh cells weretransfected with constructs encoding scFv antibodies specific for humancostimulatory molecules, CD152, CD28, CD40, and CD20, fused to humanIgG1 wild-type hinge-CH2-CH3, which was fused to human CD80transmembrane and cytoplasmic tail domains. CDC activity was measured inthe presence and absence of rabbit complement (plus C′ and no C′,respectively). The data represent the average of duplicate samples. Rehanti-hCD 152 scFvIg: Reh cells transfected with polynucleotide 10A8 scFvIgG MTH (SSS) MT CH2CH3 (SEQ ID NOs: 270 and 485); Reh anti-hCD28scFvIg:2E12 scFv IgG MTH (SSS) MT CH2CH3 (SEQ ID NOs: 268 and 483); Rehanti-hCD40scFvIg: 4.2.220 scFv IgG MTH (SSS) MT CH2CH3 (SEQ ID NOs: 266and 481); and Reh anti-hCD2OscFvIg: 2H7 scFv IgG MTH (SSS) MTCH2CH3-CD80.

FIG. 30 presents ADCC activity of Reh cells that were transfected withconstructs encoding scFv antibodies specific for human costimulatorymolecules, CD152, CD28, CD40, and CD20, as described for FIG. 29, andfor murine CD3, fused to human mutant IgG1 hinge and mutant CH2 and wildtype CH3 (Reh anti-mCD3scFv designating Reh cells transfected withpolynucleotide 500A2 scFv IgG MTH (SSS) MTCH2WTCH3 SEQ ID NO: 272 and487)), which was fused to human CD80 transmembrane and cytoplasmic taildomains. The data represent the average of quadruplicate samples.

FIG. 31 lists immunoglobulin constant region constructs that were usedin experiments illustrated in subsequent figures.

FIG. 32 depicts CDC activity of CTLA-4 Ig fusion proteins, CTLA-4 IgGWTH (CCC) WTCH2CH3 (SEQ ID NO: 307) (2 μg/ml) and CTLA-4 IgG MTHMTCH2WTCH3 (SEQ ID NO: 316 and 530) (2 μg/ml), in the presence andabsence of rabbit complement (plus C′ and no C′, respectively). Thetarget cells were Reh cells and Reh cells transfected with CD80 (RehCD80.10).

FIG. 33 shows ADCC activity of CTLA-4 Ig fusion proteins, CTLA-4 IgG WTH(CCC) WTCH2CH3 (SEQ ID NO: 307) (2 μg/ml) and CTLA-4 IgG MTH MTCH2WTCH3(SEQ ID NO: 316 and 530) (2 μg/ml). Effector cells, human PBMC, wereadded to target cells, Reh or Reh CD80.1, at the ratios indicated. FIG.33A presents the level of natural killing in Reh CD80.1 cells in theabsence of any Ig fusion protein. FIG. 33B presents ADCC mediated byCTLA-4 IgG MTH MTCH2WTCH3, and FIG. 33C presents ADCC mediated by CTLA-4IgG WTH (CCC) WTCH2CH3. Each data point represents the average percentspecific killing measured in four sample wells.

FIG. 34 illustrates binding of 2H7 (anti-CD20) scFv Ig fusion proteinsto (CD20+) CHO cells by flow immunocytofluorimetry.

FIG. 35 presents an immunoblot of 2H7 scFv IgG and IgA fusion proteins.COS cells were transiently transfected with various 2H7 scFv Ig fusionprotein constructs. The expressed polypeptides were immune precipitatedwith protein A, separated in a non-reducing SDS polyacrylamide gel, andthen transferred to a polyvinyl fluoride membrane. Proteins weredetected using an anti-human IgG (Fc specific) horseradish peroxidaseconjugate. Lane 1: vector only; lane 2: 2H7 scFv IgG WTH (CCC) WTCH2CH3(SEQ ID NO: 15 and 240); lane 3: 2H7 scFv IgG MTH (CSS) WTCH2CH3 (SEQ IDNO: 670); lane 4: 2H7 scFv IgG MTH (SCS) WTCH2CH3 (SEQ ID NO: 671); lane5: 2H7 scFv IgAH IgG WTCH2CH3 (SEQ ID NO: 18 and 284); and lane 6: 2H7scFv IgG MTH (SSS) WTCH2CH3 (SEQ ID NO: 17, 274 and 489).

FIG. 36 illustrates binding of 2H7 scFv IgAH IgACH2CH3 polypeptide (SEQID NO: 286 and 502) and 2H7 scFv IgAH IgAT4 (SEQ ID NO: 299) to (CD20+)CHO cells by flow immunocytofluorimetry. The source of the polypeptideswas culture supernatants from transiently transfected COS cells. COScells transfected with a plasmid comprising a sequence encoding 2H7 scFvIgAH IgACH2CH3 were co-transfected with a plasmid containing nucleotidesequence encoding human J chain.

FIG. 37 illustrates ADCC activity of anti-CD20 (2H7) scFv Ig fusionproteins against BJAB target cells using whole blood as the source ofeffector cells. Purified 2H7 scFv Ig fusion proteins were titrated andcombined with ⁵¹Cr-labeled BJAB cells (5×10⁴) and whole blood (1:4 finaldilution). Each data point represents the average percent specifickilling measured in four sample wells.

FIG. 38 demonstrates ADCC activity of 2H7 scFv Ig fusion proteins (5μg/ml) against ⁵¹Cr-labeled BJAB cells at 0.25, 0.125, and 0.625dilutions of whole blood. Each data point represents the average percentspecific killing measured in four sample wells.

FIG. 39 shows a comparison of ADCC activity of 2H7 scFv IgG MTH (SSS)WTCH2CH3 (5 μg/ml) and 2H7 scFv IgAH IgACH2CH3 (5 μg/ml) when human PBMCare the source of effector cells (FIG. 39A) and when human whole bloodis the source of effector cells (FIG. 39B).

FIG. 40 presents an immunoblot of 2H7 scFv IgG fusion proteins. COScells were transiently transfected with various 2H7 scFv Ig fusionprotein constructs. Culture supernatants containing the expressedpolypeptides were separated in a non-reducing SDS polyacrylamide gel,and then were transferred to a polyvinyl fluoride membrane. Proteinswere detected using an anti-human IgG (Fc specific) horseradishperoxidase conjugate. Lanes 1-5: purified 2H7 scFv IgG MTH (SSS)WTCH2CH3 at 40 ng, 20 ng, 10 ng/5 ng, and 2.5 ng per lane, respectively.Culture supernatants were separated in lanes 6-9. Lane 6: 2H7 scFv IgGWTH (CCC) WTCH2CH3; lane 7: 2H7 scFv IgG MTH (CSS) WTCH2CH3; lane 8: 2H7scFv IgG MTH (SCS) WTCH2CH3; and lane 9: 2H7 scFv VHSER11 IgG MTH (SSS)WTCH2CH3. The molecular weight (kDal) of marker proteins is indicated onthe left side of the immunoblot.

FIG. 41A illustrates cell surface expression of 1D8 (anti-murine 4-1BB)scFv IgG WTH WTCH2CH3-CD80 fusion protein on K1735 melanoma cells byflow immunofluorimetry. The scFv fusion protein was detected withphycoerythrin-conjugated F(ab′)₂ goat anti-human IgG. FIG. 41B depictsgrowth of tumors in naïve C3H mice transplanted by subcutaneousinjection with wild type K1735 melanoma cells (K1735-WT) or with K1735cells transfected with 1D8 scFv IgG WTH WTCH2CH3-CD80 (K1735-1D8). Tumorgrowth was monitored by measuring the size of the tumor. FIG. 41Cdemonstrates the kinetics of tumor growth in naïve C3H mice injectedintraperitoneally with monoclonal antibodies to remove CD8+, CD4+, orboth CD4+ and CD8+ T cells prior to transplantation of the animals withK1735-1D8 cells.

FIG. 42 demonstrates therapy of established K1735-WT tumors usingK1735-1D8 as an immunogen. Six days after mice were transplanted withK1735-WT tumors, one group (five animals) was injected subcutaneouslywith K1735-1D8 cells (open circles) or irradiated K1735-WT cells (solidsquares) on the contralateral side. A control group of mice received PBS(open squares). Treatments were repeated on the days indicated by thearrows.

FIG. 43 shows the growth of tumors in animals that were injectedsubcutaneously with 2×10⁶ K1735-WT cells (solid squares) and the growthof tumors in animals that were injected subcutaneously with 2×10⁶K1735-WT cells plus 2×10⁵ K1735-1D8 cells (open triangles).

FIG. 44 presents a flow cytometry analysis of Ag104 murine sarcoma tumorcells transfected with 1D8 scFv IgG WTH WTCH2CH3-CD80 isolated afterrepeated rounds of panning against anti-human IgG. Transfected cellsexpressing 1D8 scFv IgG WTH WTCH2CH3-CD80 were detected withfluoroisothiocyanate (FITC)-conjugated goat anti-human IgG (depicted inblack). Untransfected cells are shown in gray.

FIG. 45 illustrates migration of various 2H7 scFv Ig fusion proteins ina 10% SDS-PAGE gel. 2H7 was the anti-CD20 scFv and [220] was theanti-CD40 scFv. Lane 1: Bio-Rad prestained molecular weight standards;lane 2: anti-CD20 scFv IgG MTH (SSS) MTCH2WTCH3; lane 3: anti-CD20 scFvIgG MTH (SSS) WTCH2CH3; lane 4: 2H7 scFv IgAH IgG WTCH2CH3; lane 5:anti-CD20-anti-CD40 scFv IgG MTH (SSS) MTCH2WTCH3; lane 6: Rituximab;lane 7: Novex Multimark® molecular weight standards.

FIG. 46 illustrates effector function as measured in an ADCC assay of2H7 Ig fusion proteins that contain a mutant CH2 domain or wild type CH2domain. The percent specific killing of BJAB target cells in thepresence of human PBMC effector cells by 2H7 scFv IgG MTH (SSS)MTCH2WTCH3 (diamonds) was compared to 2H7 scFv IgG MTH (SSS) WTCH2CH3(squares) and 2H7 scFv IgAH IgG WTCH2CH3 (triangles) and Rituximab(circles).

FIG. 47 shows cell surface expression of an anti-human CD3 scFv IgG WTHWTCH2CH3-CD80 (SEQ ID NO: 560) fusion protein on Reh cells (FIG. 47A)and T51 lymphoblastoid cells (FIG. 47B) by flow immunocytofluorimetry.

FIG. 48 presents the percent specific killing of untransfected Reh andT51 cells and the percent specific killing of Reh cells (Reh anti-hCD3)(FIG. 48A) and T51 cells (T51 anti-hCD3) (FIG. 48B) that weretransfected with a construct encoding scFv antibodies specific for humanCD3, fused to human IgG1 wild-type hinge-CH2-CH3, which was fused tohuman CD80 transmembrane and cytoplasmic tail domains (anti-human CD3scFv IgG WTH WTCH2CH3-CD80 (SEQ ID NO: 560). Human PBMC (effector cells)were combined with BJAB target cells at the ratios indicated.

FIG. 49 illustrates binding of 5B9, a murine anti-human CD137 (4-1BB)monoclonal antibody, and a 5B9 scFv IgG fusion protein (5B9 scFv IgG MTH(SSS) WTCH2CH3 (SEQ ID NO: 363)) to stimulated human PBMC. Binding ofthe 5B9 scFv IgG fusion protein was detected by flowimmunocytofluorimetry using FITC conjugated goat anti-human IgG. Bindingof the 5B9 monoclonal antibody was detected with FITC conjugated goatanti-mouse IgG.

DETAILED DESCRIPTION

The present invention is directed to binding domain-immunoglobulinfusion proteins and to related compositions and methods, which will beuseful in immunotherapeutic and immunodiagnostic applications, and whichoffer certain advantages over antigen-specific polypeptides of the priorart. The fusion proteins of the present invention are preferably singlepolypeptide chains that comprise, in pertinent part, the following fuseddomains: a binding domain polypeptide, an immunoglobulin hinge regionpolypeptide, an immunoglobulin heavy chain CH2 constant regionpolypeptide, and an immunoglobulin heavy chain CH3 constant regionpolypeptide. According to certain preferred embodiments the fusionproteins of the present invention further comprise a plasma membraneanchor domain. According to certain other preferred embodiments thefusion proteins of the present invention further comprise animmunoglobulin heavy chain CH4 constant region polypeptide. Inparticularly preferred embodiments, the polypeptide domains of which thebinding domain-immunoglobulin fusion protein is comprised are, or arederived from, polypeptides that are the products of human genesequences, but the invention need not be so limited and may in factrelate to binding domain-immunoglobulin fusion proteins as providedherein that are derived from any natural or artificial source, includinggenetically engineered and/or mutated polypeptides.

The present invention relates in part to the surprising observation thatthe binding domain-immunoglobulin fusion proteins described herein arecapable of immunological activity. More specifically, these proteinsretain the ability to participate in well known immunological effectoractivities including antibody dependent cell mediated cytotoxicity(ADCC, e.g., subsequent to antigen binding on a cell surface, engagementand induction of cytotoxic effector cells bearing appropriate Fcreceptors, such as natural killer (NK) cells bearing FcRγIII, underappropriate conditions;) and/or complement fixation in complementdependent cytotoxicity (CDC, e.g., subsequent to antigen binding on acell surface, recruitment and activation of cytolytic proteins that arecomponents of the blood complement cascade; for reviews of ADCC and CDCsee, e.g., Carter, 2001 Nat. Rev. Canc. 1:118; Sulica et al., 2001 Int.Rev. Immunol. 20:371; Maloney et al., 2002 Semin. Oncol. 29:2; Sondel etal., 2001; Maloney 2001 Anticanc. Drugs 12 Suppl. 2:1-4; IgA activationof complement by the alternative pathway is described, for example, inSchneiderman et al., 1990 J. Immunol. 145:233), despite havingstructures that would not be expected to be capable of promoting sucheffector activities. As described in greater detail below, ADCC and CDCare unexpected functions for fusion proteins comprising immunoglobulinheavy chain regions and having the structures described herein, and inparticular for immunoglobulin fusion proteins comprising immunoglobulinhinge region polypeptides that are compromised in their ability to forminterchain, homodimeric disulfide bonds.

Another advantage afforded by the present invention is a bindingdomain-immunoglobulin fusion polypeptide that can be produced insubstantial quantities that are typically greater than those routinelyattained with single-chain antibody constructs of the prior art. Inpreferred embodiments, the binding domain-immunoglobulin fusionpolypeptides of the present invention are recombinantly expressed inmammalian expression systems, which offer the advantage of providingpolypeptides that are stable in vivo (e.g., under physiologicalconditions). According to non-limiting theory, such stability may derivein part from posttranslational modifications, and specificallyglycosylation, of the fusion proteins. Production of the present bindingdomain-immunoglobulin fusion proteins via recombinant mammalianexpression has been attained in static cell cultures at a level ofgreater than 50 mg protein per liter culture supernatant and has beenroutinely observed in such cultures at 10-50 mg/l, such that preferablyat least 10-50 mg/l may be produced under static culture conditions;also contemplated are enhanced production of the fusion proteins usingart-accepted scale-up methodologies such as “fed batch” (i.e.,non-static) production, where yields of at least 5-500 mg/l, and in someinstances at least 0.5-1 gm/l, depending on the particular proteinproduct, are obtained.

A binding domain polypeptide according to the present invention may beany polypeptide that possesses the ability to specifically recognize andbind to a cognate biological molecule or complex of more than onemolecule or assembly or aggregate, whether stable or transient, of sucha molecule, which includes a protein, polypeptide, peptide, amino acid,or derivative thereof; a lipid, fatty acid or the like, or derivativethereof; a carbohydrate, saccharide or the like or derivative thereof, anucleic acid, nucleotide, nucleoside, purine, pyrimidine or relatedmolecule, or derivative thereof, or the like; or any combination thereofsuch as, for example, a glycoprotein, a glycopeptide, a glycolipid, alipoprotein, a proteolipid; or any other biological molecule that may bepresent in a biological sample. Biological samples may be provided byobtaining a blood sample, biopsy specimen, tissue explant, organculture, biological fluid or any other tissue or cell preparation from asubject or a biological source. The subject or biological source may bea human or non-human animal, a primary cell culture or culture adaptedcell line including but not limited to genetically engineered cell linesthat may contain chromosomally integrated or episomal recombinantnucleic acid sequences, immortalized or immortalizable cell lines,somatic cell hybrid cell lines, differentiated or differentiatable celllines, transformed cell lines and the like. In certain preferredembodiments of the invention, the subject or biological source may besuspected of having or being at risk for having a malignant condition ora B-cell disorder as provided herein, which in certain further preferredembodiments may be an autoimmune disease, and in certain other preferredembodiments of the invention the subject or biological source may beknown to be free of a risk or presence of such disease.

A binding domain polypeptide may therefore be any naturally occurring orrecombinantly produced binding partner for a cognate biological moleculeas provided herein that is a target structure of interest, hereinreferred to as an “antigen” but intended according to the presentdisclosure to encompass any target biological molecule to which it isdesirable to have the subject invention fusion protein specificallybind. Binding domain-immunoglobulin fusion proteins are defined to be“immunospecific” or capable of specifically binding if they bind adesired target molecule such as an antigen as provided herein, with aK_(a) of greater than or equal to about 10⁴ M⁻¹, preferably of greaterthan or equal to about 10⁵ M⁻¹, more preferably of greater than or equalto about 10⁶ M⁻¹ and still more preferably of greater than or equal toabout 10⁷ M⁻¹. Affinities of binding domain-immunoglobulin fusionproteins according to the present invention can be readily determinedusing conventional techniques, for example those described by Scatchardet al., Ann. N.Y. Acad. Sci. 51:660 (1949). Such determination of fusionprotein binding to target antigens of interest can also be performedusing any of a number of known methods for identifying and obtainingproteins that specifically interact with other proteins or polypeptides,for example, a yeast two-hybrid screening system such as that describedin U.S. Pat. No. 5,283,173 and U.S. Pat. No. 5,468,614, or theequivalent.

Preferred embodiments of the subject invention bindingdomain-immunoglobulin fusion protein comprise binding domains thatinclude at least one immunoglobulin variable region polypeptide, such asall or a portion or fragment of a heavy chain or a light chain V-region,provided it is capable of specifically binding an antigen or otherdesired target structure of interest as described herein. In otherpreferred embodiments the binding domain comprises a single chainimmunoglobulin-derived Fv product, which may include all or a portion ofat least one immunoglobulin light chain V-region and all or a portion ofat least one immunoglobulin heavy chain V-region, and which furthercomprises a linker fused to the V-regions; preparation and testing suchconstructs are described in greater detail herein and are well known inthe art. As described herein and as also known in the art,immunoglobulins comprise products of a gene family the members of whichexhibit a high degree of sequence conservation, such that amino acidsequences of two or more immunoglobulins or immunoglobulin domains orregions or portions thereof (e.g., VH domains, VL domains, hingeregions, CH2 constant regions, CH3 constant regions) can be aligned andanalyzed to identify portions of such sequences that correspond to oneanother, for instance, by exhibiting pronounced sequence homology.Determination of sequence homology may be readily determined with any ofa number of sequence alignment and analysis tools, including computeralgorithms well known to those of ordinary skill in the art, such asAlign or the BLAST algorithm (Altschul, J. Mol. Biol. 219:555-565, 1991;Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992),which is available at the NCBI website(http://www/ncbi.nlm.nih.gov/cgi-bin/BLAST). Default parameters may beused.

Portions of a particular immunoglobulin reference sequence and of anyone or more additional immunoglobulin sequences of interest that may becompared to the reference sequence are regarded as “corresponding”sequences, regions, fragments or the like, based on the convention fornumbering immunoglobulin amino acid positions according to Kabat,Sequences of Proteins of Immunological Interest, (5^(th) ed. Bethesda,Md.: Public Health Service, National Institutes of Health (1991)). Forexample, according to this convention, the immunoglobulin family towhich an immunoglobulin sequence of interest belongs is determined basedon conservation of variable region polypeptide sequence invariant aminoacid residues, to identify a particular numbering system for theimmunoglobulin family, and the sequence(s) of interest can then bealigned to assign sequence position numbers to the individual aminoacids which comprise such sequence(s). Preferably at least 70%, morepreferably at least 80%-85% or 86%-89%, and still more preferably atleast 90%, 92%, 94%, 96%, 98% or 99% of the amino acids in a given aminoacid sequence of at least 1000, more preferably 700-950, more preferably350-700, still more preferably 100-350, still more preferably 80-100,70-80, 60-70, 50-60, 40-50 or 30-40 consecutive amino acids of asequence, are identical to the amino acids located at correspondingpositions in a reference sequence such as those disclosed by Kabat(1991) or in a similar compendium of related immunoglobulin sequences,such as may be generated from public databases (e.g., Genbank,SwissProt, etc.) using sequence alignment tools as described above. Incertain preferred embodiments, an immunoglobulin sequence of interest ora region, portion, derivative or fragment thereof is greater than 95%identical to a corresponding reference sequence, and in certainpreferred embodiments such a sequence of interest may differ from acorresponding reference at no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acid positions.

For example, in certain embodiments the present invention is directed toa binding domain-immunoglobulin fusion protein comprising in pertinentpart a human immunoglobulin heavy chain variable region polypeptidecomprising a mutation at an amino acid at a location corresponding toamino acid position 11 in the VH domain, or amino acid position 155 inSEQ ID NO: 11, position 158 in SEQ ID NO: 12, position 154 in SEQ IDNO:13, or position 159 in SEQ ID NO 14, or amino acid position 11 in theVH domain polypeptides listed in SEQ ID NOS: 341, 354, 465, 471, 477,548 which comprises a murine VH-derived sequence, and regarding which itis noteworthy that at a relatively limited number of immunoglobulin VHsequence positions, including position 11, amino acid conservation isobserved in the overwhelming majority of VH sequences analyzed acrossmammalian species lines (e.g., Leu11, Val37, Gly44, Leu45, Trp47; Nguyenet al., 1998 J. Mol. Biol 275:413). These amino acid side chains arelocated at the surface of the variable domain (V_(H)), where they maycontact residues of the C_(H)1 (Leu11) and the V_(L) domains (Val37,Gly44, Leu45, and Trp47) and may, in the absence of light chains,contribute to stability and solubility of the protein (see, e.g.,Chothia et al., 1985 J. Mol. Biol. 186:651; Muyldermans et al., 1994Prot. Engineer. 7:1129; Desmyter et al., 1996 Nat. Struct. Biol. 3:803;Davies et al., 1994 FEBS Lett. 339:285). As another example, byreference to immunoglobulin sequence compendia and databases such asthose cited above, the relatedness of two or more immunoglobulinsequences to each other can readily and without undue experimentation beestablished in a manner that permits identification of the animalspecies of origin, the class and subclass (e.g., isotype) of aparticular immunoglobulin or immunoglobulin region polypeptide sequence.Any immunoglobulin variable region polypeptide sequence, including VHand/or VL and/or single-chain variable region (sFv) sequences or other Vregion-derived sequences or the like, may be used as a binding domain.Preferred embodiments include immunoglobulin V region polypeptidesequences derived from monoclonal antibodies such as murine or otherrodent antibodies, or monoclonal antibodies derived from other sourcessuch as goat, rabbit, equine, bovine, camelid or other species,including transgenic animals, and also including human or humanizedmonoclonal antibodies. Non-limiting examples include variable regionpolypeptide sequences derived from mAb such as those described ingreater detail in the Examples below, for instance, CD20-specific murinemonoclonal antibodies (e.g., 2H7), mAb L6 (specific for acarbohydrate-defined epitope and available from American Type CultureCollection, Manassas, Va., as hybridoma HB8677), and mAb specific forCD28 (e.g., mAb 2E12), CD40, CD80, CD137 (e.g., mAb 5B9 or mAb 1D8 whichrecognizes the human homologue of CD137, 41BB) and CD152 (CTLA-4).

Other binding domain polypeptides may comprise any protein or portionthereof that retains the ability to specifically bind an antigen asprovided herein, including non-immunoglobulins. Accordingly theinvention contemplates fusion proteins comprising binding domainpolypeptides that are derived from polypeptide ligands such as hormones,cytokines, chemokines, and the like; cell surface or soluble receptorsfor such polypeptide ligands; lectins; intercellular adhesion receptorssuch as specific leukocyte integrins, selectins, immunoglobulin genesuperfamily members, intercellular adhesion molecules (ICAM-1, -2, -3)and the like; histocompatibility antigens; etc.

Examples of cell surface receptors that may provide a binding domainpolypeptide, and that may also be selected as the target molecule orantigen to which a binding domain-Ig fusion protein of the presentinvention desirably binds, include the following, or the like: HER1(e.g., GenBank Accession Nos. U48722, SEG_HEGFREXS, KO3193), HER2(Yoshino et al., 1994 J. Immunol. 152:2393; Disis et al., 1994 Canc.Res. 54:16; see also, e.g., GenBank Acc. Nos. X03363, M17730,SEG_HUMHER20), HER3 (e.g., GenBank Acc. Nos. U29339, M34309), HER4(Plowman et al., 1993 Nature 366:473; see also e.g., GenBank Acc. Nos.L07868, T64105), epidermal growth factor receptor (EGFR) (e.g., GenBankAcc. Nos. U48722, SEG_HEGFREXS, KO3193), vascular endothelial cellgrowth factor (e.g., GenBank No. M32977), vascular endothelial cellgrowth factor receptor (e.g., GenBank Acc. Nos. AF022375, 1680143,U48801, X62568), insulin-like growth factor-I (e.g., GenBank Acc. Nos.X00173, X56774, X56773, X06043, see also European Patent No. GB2241703), insulin-like growth factor-II (e.g., GenBank Acc. Nos. X03562,X00910, SEG_HUMGFIA, SEG HUMGFI2, M17863, M17862), transferrin receptor(Trowbridge and Omary, 1981 Proc. Nat. Acad. USA 78:3039; see also e.g.,GenBank Acc. Nos. X01060, M11507), estrogen receptor (e.g., GenBank Acc.Nos. M38651, X03635, X99101, U47678, M12674), progesterone receptor(e.g., GenBank Acc. Nos. X51730, X69068, M15716), follicle stimulatinghormone receptor (FSH-R) (e.g., GenBank Acc. Nos. Z34260, M65085),retinoic acid receptor (e.g., GenBank Acc. Nos. L12060, M60909, X77664,X57280, X07282, X06538), MUC-1 (Barnes et al., 1989 Proc. Nat. Acad.Sci. USA 86:7159; see also e.g., GenBank Acc. Nos. SEG_MUSMUCIO, M65132,M64928) NY-ESO-1 (e.g., GenBank Acc. Nos. AJ003149, U87459), NA 17-A(e.g., European Patent No. WO 96/40039), Melan-A/MART-1 (Kawakami etal., 1994 Proc. Nat. Acad. Sci. USA 91:3515; see also e.g., GenBank Acc.Nos. U06654, U06452), tyrosinase (Topalian et al., 1994 Proc. Nat. Acad.Sci. USA 91:9461; see also e.g., GenBank Acc. Nos. M26729, SEG_HUMTYR0,see also Weber et al., J. Clin. Invest (1998) 102:1258), Gp-100(Kawakami et al., 1994 Proc. Nat. Acad. Sci. USA 91:3515; see also e.g.,GenBank Acc. No. 573003, see also European Patent No. EP 668350; Ademaet al., 1994 J. Biol. Chem. 269:20126), MAGE (van den Bruggen et al.,1991 Science 254:1643; see also, e.g., GenBank Acc. Nos. U93163,AF064589, U66083, D32077, D32076, D32075, U10694, U10693, U10691,U10690, U10689, U10688, U10687, U10686, U10685, L18877, U10340, U10339,L18920, U03735, M77481), BAGE (e.g., GenBank Acc. No. U19180, see alsoU.S. Pat. Nos. 5,683,886 and 5,571,711), GAGE (e.g., GenBank Acc. Nos.AF055475, AF055474, AF055473, U19147, U19146, U19145, U19144, U19143,U19142), any of the CTA class of receptors including in particularHOM-MEL-40 antigen encoded by the SSX2 gene (e.g., GenBank Acc. Nos.X86175, U90842, U90841, X86174), carcinoembyonic antigen (CEA, Gold andFreedman, 1985 J. Exp. Med. 121:439; see also e.g., GenBank Acc. Nos.SEG_HUMCEA, M59710, M59255, M29540), and PyLT (e.g., GenBank Acc. Nos.J02289, J02038).

Additional cell surface receptors that may be sources of binding domainpolypeptides or that may be cognate antigens include the following, orthe like: CD2 (e.g., GenBank Acc. Nos. Y00023, SEG_HUMCD2, M16336,M16445, SEG_MUSCD2, M14362), 4-1BB (CDw137, Kwon et al., 1989 Proc. Nat.Acad. Sci. USA 86:1963, 4-1BB ligand (Goodwin et al., 1993 Eur. J.Immunol. 23:2361; Melero et al., 1998 Eur. J. Immunol. 3:116), CD5(e.g., GenBank Acc. Nos. X78985, X89405), CD10 (e.g., GenBank Acc. Nos.M81591, X76732) CD27 (e.g., GenBank Acc. Nos. M63928, L24495, L08096),CD28 (June et al., 1990 Immunol. Today 11:211; see also, e.g., GenBankAcc. Nos. J02988, SEG_HUMCD28, M34563), CD152/CTLA-4 (e.g., GenBank Acc.Nos. L15006, X05719, SEG_HUMIGCTL), CD40 (e.g., GenBank Acc. Nos.M83312, SEG_MUSC040A0, Y10507, X67878, X96710, U15637, L07414),interferon-γ (IFN-γ; see, e.g., Farrar et al. 1993 Ann. Rev. Immunol.11:571 and references cited therein, Gray et al. 1982 Nature 295:503,Rinderknecht et al. 1984 J. Biol. Chem. 259:6790, DeGrado et al. 1982Nature 300:379), interleukin-4 (IL-4; see, e.g., 53^(rd) Forum inImmunology, 1993 Research in Immunol. 144:553-643; Banchereau et al.,1994 in The Cytokine Handbook, 2^(nd) ed., A. Thomson, ed., AcademicPress, NY, p. 99; Keegan et al., 1994 J Leukocyt. Biol.. 55:272, andreferences cited therein), interleukin-17 (IL-17) (e.g., GenBank Acc.Nos. U32659, U43088) and interleukin-17 receptor (IL-17R) (e.g., GenBankAcc. Nos. U31993, U58917). Notwithstanding the foregoing, the presentinvention expressly does not encompass any immunoglobulin fusion proteinthat is disclosed in U.S. Pat. No. 5,807,734, U.S. Pat. No. 5,795,572 orU.S. Pat. No. 5,807,734.

Additional cell surface receptors that may be sources of binding domainpolypeptides or that may be cognate antigens include the following, orthe like: CD59 (e.g., GenBank Acc. Nos. SEG_HUMCD590, M95708, M34671),CD48 (e.g., GenBank Acc. Nos. M59904), CD58/LFA-3 (e.g., GenBank Acc.No. A25933, Y00636, E12817; see also JP 1997075090-A), CD72 (e.g.,GenBank Acc. Nos. AA311036, 540777, L35772), CD70 (e.g., GenBank Acc.Nos. Y13636, S69339), CD80/B7.1 (Freeman et al., 1989 J. Immunol.43:2714; Freeman et al., 1991 J. Exp. Med. 174:625; see also e.g.,GenBank Acc. Nos. U33208, 1683379), CD86/B7.2 (Freeman et al., 1993 J.Exp. Med. 178:2185, Boriello et al., 1995 J. Immunol. 155:5490; seealso, e.g., GenBank Acc. Nos. AF099105, SEG_MMB72G, U39466, U04343,SEG_HSB725, L25606, L25259), B7-H1/B7-DC (e.g., Genbank Acc. Nos.NM_(—)014143, AF177937, AF317088; Dong et al., 2002 Nat. Med. June 24[epub ahead of print], PMID 12091876; Tseng et al., 2001 J. Exp. Med.193:839; Tamura et al., 2001 Blood 97:1809; Dong et al., 1999 Nat. Med.5:1365), CD40 ligand (e.g., GenBank Acc. Nos. SEG_HUMCD40L, X67878,X65453, L07414), IL-17 (e.g., GenBank Acc. Nos. U32659, U43088), CD43(e.g., GenBank Acc. Nos. X52075, J04536), ICOS (e.g., Genbank Acc. No.AH011568), CD3 (e.g., Genbank Acc. Nos. NM_(—)000073 (gamma subunit),NM_(—)000733 (epsilon subunit), X73617 (delta subunit)), CD4 (e.g.,Genbank Acc. No. NM_(—)000616), CD25 (e.g., Genbank Acc. No.NM_(—)000417), CD8 (e.g., Genbank Acc. No. M12828), CD11b (e.g., GenbankAcc. No. J03925), CD14 (e.g., Genbank Acc. No. XM_(—)039364), CD56(e.g., Genbank Acc. No. U63041), CD69 (e.g., Genbank Acc. No.NM_(—)001781) and VLA-4 (α₄β₇) (e.g., GenBank Acc. Nos. L12002, X16983,L20788, U97031, L24913, M68892, M95632). The following cell surfacereceptors are typically associated with B cells: CD19 (e.g., GenBankAcc. Nos. SEG_HUMCD19W0, M84371, SEG MUSCD19W, M62542), CD20 (e.g.,GenBank Acc. Nos. SEG_HUMCD20, M62541), CD22 (e.g., GenBank Acc. Nos.1680629, Y10210, X59350, U62631, X52782, L16928), CD30 (e.g., GenbankAcc. Nos. M83554, D86042), CD153 (CD30 ligand, e.g., GenBank Acc. Nos.L09753, M83554), CD37 (e.g., GenBank Acc. Nos. SEG_MMCD37X, X14046,X53517), LD50 (ICAM-3, e.g., GenBank Acc. No. NM_(—)002162), CD106(VCAM-1) (e.g., GenBank Acc. Nos. X53051, X67783, SEG_MMVCAM1C, see alsoU.S. Pat. No. 5,596,090), CD54 (ICAM-1) (e.g., GenBank Acc. Nos. X84737,582847, X06990, J03132, SEG_MUSICAM0), interleukin-12 (see, e.g., Reiteret al, 1993 Crit. Rev. Immunol. 13:1, and references cited therein),CD134 (OX40, e.g., GenBank Acc. No. AJ277151), CD137 (41BB, e.g.,GenBank Acc. No. L12964, NM_(—)001561), CD83 (e.g., GenBank Acc. Nos.AF001036, AL021918), DEC-205 (e.g., GenBank Acc. Nos. AF011333, U19271).

Binding domain-immunoglobulin fusion proteins of the present inventioncomprise a binding domain polypeptide that, according to certainparticularly preferred embodiments, is capable of specifically bindingat least one antigen that is present on an immune effector cell.According to non-limiting theory, such binding domain-Ig fusion proteinsmay advantageously recruit desired immune effector cell function(s) in atherapeutic context, where it is well known that immune effector cellshaving different specialized immune functions can be distinguished fromone another on the basis of their differential expression of a widevariety of cell surface antigens, such as many of the antigens describedherein to which binding domain polypeptides can specifically bind.Immune effector cells include any cell that is capable of directlymediating an activity which is a component of immune system function,including cells having such capability naturally or as a result ofgenetic engineering.

In certain embodiments an immune effector cell comprises a cell surfacereceptor for an immunoglobulin, such as a receptor for an immunoglobulinconstant region and including the class of receptors commonly referredto as “Fc receptors” (FcR). A number of FcR have been structurallyand/or functionally characterized and are well known in the art,including FcR having specific abilities to interact with a restrictedsubset of immunoglobulin heavy chain isotypes, or that interact with Fcdomains with varying affinities, and/or which may be expressed onrestricted subsets of immune effector cells under certain conditions(e.g., Kijimoto-Ochichai et al., 2002 Cell Mol. Life Sci. 59:648; Daviset al., 2002 Curr. Top. Microbiol. Immunol. 266:85; Pawankar, 2001 Curr.Opin. Allerg. Clin. Immunol. 1:3; Radaev et al., 2002 Mol. Immunol.38:1073; Wurzburg et al., 2002 Mol. Immunol. 38:1063; Sulica et al.,2001 Int. Rev. Immunol. 20:371; Underhill et al., 2002 Ann. Rev.Immunol. 20:825; Coggeshall, 2002 Curr. Dir. Autoimm. 5:1; Mimura etal., 2001 Adv. Exp. Med. Biol. 495:49; Baumann et al., 2001 Adv. Exp.Med. Biol. 495:219; Santoso et al., 2001 Ital. Heart J. 2:811; Novak etal., 2001 Curr. Opin. Immunol. 13:721; Fossati et al., 2001 Eur. J.Clin. Invest. 31:821).

Cells that are capable of mediating ADCC are preferred examples ofimmune effector cells according to the present invention. Otherpreferred examples include natural killer (NK) cells, tumor-infiltratingT lymphocytes (TIL), cytotoxic T lymphocytes (CTL), and granulocyticcells such as cells that comprise allergic response mechanisms. Immuneeffector cells thus include, but are not limited to, cells ofhematopoietic origins including cells at various stages ofdifferentiation within myeloid and lymphoid lineages and which may (butneed not) express one or more types of functional cell surface FcR, suchas T lymphocytes, B lymphocytes, NK cells, monocytes, macrophages,dendritic cells, neutrophils, basophils, eosinophils, mast cells,platelets, erythrocytes, and precursors, progenitors (e.g.,hematopoietic stem cells), quiescent, activated and mature forms of suchcells. Other immune effector cells may include cells ofnon-hematopoietic origin that are capable of mediating immune functions,for example, endothelial cells, keratinocytes, fibroblasts, osteoclasts,epithelial cells and other cells. Immune effector cells may also includecells that mediate cytotoxic or cytostatic events, or endocytic,phagocytic, or pinocytotic events, or that effect induction ofapoptosis, or that effect microbial immunity or neutralization ofmicrobial infection, or cells that mediate allergic, inflammatory,hypersensitivity and/or autoimmune reactions.

Allergic response mechanisms are well known in the art and include anantigen (e.g., allergen)-specific component such as an immunoglobulin(e.g., IgE), as well as the cells and mediators which comprise sequelaeto allergen-immunoglobulin (e.g., IgE) encounters (e.g., Ott et al.,2000 J. Allerg. Clin. Immunol. 106:429; Barnes, 2000 J. Allerg. Clin.Immunol. 106:5; Togias, 2000 J. Allerg. Clin. Immunol. 105:S599; Akdiset al., 2000 Int. Arch. Allerg. Immunol. 121:261; Beach, 2000 Occup.Med. 15:455). Particularly with regard to binding domain-immunoglobulinfusion proteins of the present invention that interact with FcR, certainembodiments of the present invention contemplate fusion proteins thatcomprise one or more IgE-derived domains and that are capable ofinducing an allergic response mechanism that comprises IgE-specific FcR,as also noted above and as described in the cited references. Withoutwishing to be bound by theory, and as disclosed herein, fusion proteinsof the present invention may comprise portions of IgE heavy chain Fcdomain polypeptides, whether expressed as cell surface proteins (e.g.,with a plasma membrane anchor domain) or as soluble proteins (e.g.,without a plasma membrane anchor domain). Further according tonon-limiting theory, recruitment and induction of an allergic responsemechanism (e.g., an FcR-epsilon expressing immune effector cell) mayproceed as the result of either or both of the presence of an IgE Fcdomain (e.g., that is capable of triggering an allergic mechanism by FcRcrosslinking) and the presence of the cognate antigen to which thebinding domain specifically binds. The present invention thereforeexploits induction of allergic response mechanisms in heretoforeunappreciated contexts, such as treatment of a malignant condition or aB-cell disorder as described herein.

An immunoglobulin hinge region polypeptide, as discussed above, includesany hinge peptide or polypeptide that occurs naturally, as an artificialpeptide or as the result of genetic engineering and that is situated inan immunoglobulin heavy chain polypeptide between the amino acidresidues responsible for forming intrachain immunoglobulin-domaindisulfide bonds in CH1 and CH2 regions; hinge region polypeptides foruse in the present invention may also include a mutated hinge regionpolypeptide. Accordingly, an immunoglobulin hinge region polypeptide maybe derived from, or may be a portion or fragment of (i.e., one or moreamino acids in peptide linkage, typically 15-115 amino acids, preferably95-110, 80-94, 60-80, or 5-65 amino acids, preferably 10-50, morepreferably 15-35, still more preferably 18-32, still more preferably20-30, still more preferably 21, 22, 23, 24, 25, 26, 27, 28 or 29 aminoacids) an immunoglobulin polypeptide chain region classically regardedas having hinge function, as described above, but a hinge regionpolypeptide for use in the instant invention need not be so restrictedand may include amino acids situated (according to structural criteriafor assigning a particular residue to a particular domain that may vary,as known in the art) in an adjoining immunoglobulin domain such as a CH1domain or a CH2 domain, or in the case of certain artificiallyengineered immunoglobulin constructs, an immunoglobulin variable regiondomain.

Wild-type immunoglobulin hinge region polypeptides include any naturallyoccurring hinge region that is located between the constant regiondomains, CH1 and CH2, of an immunoglobulin. The wild-type immunoglobulinhinge region polypeptide is preferably a human immunoglobulin hingeregion polypeptide, preferably comprising a hinge region from a humanIgG, IgA or IgE immunoglobulin, and more preferably, a hinge regionpolypeptide from a wild-type or mutated human IgG1 isotype as describedherein. As is known to the art, despite the tremendous overall diversityin immunoglobulin amino acid sequences, immunoglobulin primary structureexhibits a high degree of sequence conservation in particular portionsof immunoglobulin polypeptide chains, notably with regard to theoccurrence of cysteine residues which, by virtue of their sulfhydrylgroups, offer the potential for disulfide bond formation with otheravailable sulfhydryl groups.

Accordingly, in the context of the present invention wild-typeimmunoglobulin hinge region polypeptides may be regarded as those thatfeature one or more highly conserved (e.g., prevalent in a population ina statistically significant manner) cysteine residues, and in certainpreferred embodiments a mutated hinge region polypeptide may be selectedthat contains zero or one cysteine residue and that is derived from sucha wild-type hinge region.

In certain preferred embodiments wherein the hinge region polypeptide isa mutated human IgG1 immunoglobulin hinge region polypeptide that isderived from a wild-type hinge region, it is noted that the wild-typehuman IgG1 hinge region polypeptide sequence comprises threenon-adjacent cysteine residues, referred to as a first cysteine of thewild-type hinge region, a second cysteine of the wild-type hinge regionand a third cysteine of the wild-type hinge region, respectively,proceeding along the hinge region sequence from the polypeptideN-terminus toward the C-terminus. Accordingly, in certain suchembodiments of the present invention, the mutated human IgG1immunoglobulin hinge region polypeptide contains two cysteine residuesand the first cysteine of the wild-type hinge region is not mutated. Incertain other embodiments of the present invention the mutated humanIgG1 immunoglobulin hinge region polypeptide contains no more than onecysteine residue, and in certain other embodiments the mutated humanIgG1 immunoglobulin hinge region polypeptide contains no cysteineresidues.

The binding domain-immunoglobulin fusion proteins of the presentinvention expressly do not contemplate any fusion protein that isdisclosed in U.S. Pat. No. 5,892,019. For example, and as disclosed inU.S. Pat. No. 5,892,019, a mutated human IgG1 hinge region describedtherein has a substitution or deletion of the first IgG1 hinge regioncysteine residue, but retains both of the second and third IgG1 hingeregion cysteine residues that correspond to the second and thirdcysteines of the wild-type IgG1 hinge region sequence. This referencediscloses that the first cysteine residue of the wild-type IgG1 hingeregion is replaced to prevent interference, by the first cysteineresidue, with proper assembly of the single chain immunoglobulin-likepolypeptide described therein into an immunoglobulin-like dimer. As alsodisclosed in this reference, the second and third cysteines of the IgG1hinge region are retained to provide interchain disulfide linkagebetween two heavy chain constant regions to promote dimer formation,which further according to U.S. Pat. No. 5,892,019 results in animmunoglobulin-like dimer having effector function such as ADCCcapability.

By contrast and as described herein, the binding domain-immunogloblinfusion proteins of the present invention, which are capable of ADCC, arenot so limited and may comprise, in pertinent part, (i) a wild-typehuman IgG1 immunoglobulin hinge region polypeptide, (ii) a mutated humanIgG1 immunoglobulin hinge region polypeptide that is derived from awild-type immunoglobulin hinge region polypeptide having three or morecysteine residues, wherein the mutated human IgG1 immunoglobulin hingeregion polypeptide contains two cysteine residues and wherein a firstcysteine of the wild-type hinge region is not mutated, (iii) a mutatedhuman IgG1 immunoglobulin hinge region polypeptide that is derived froma wild-type immunoglobulin hinge region polypeptide having three or morecysteine residues, wherein the mutated human IgG1 immunoglobulin hingeregion polypeptide contains no more than one cysteine residue, or (iv) amutated human IgG1 immunoglobulin hinge region polypeptide that isderived from a wild-type immunoglobulin hinge region polypeptide havingthree or more cysteine residues, wherein the mutated human IgG1immunoglobulin hinge region polypeptide contains no cysteine residues.In particular, the present invention thus offers unexpected advantagesassociated with retention by the fusion proteins described herein of theability to mediate ADCC even where the ability to dimerize via IgG1hinge region interchain disulfide bonds is ablated or compromised by theremoval or replacement of one, two or three hinge region cysteineresidues, and even where the first cysteine of the IgG1 hinge region isnot mutated.

A mutated immunoglobulin hinge region polypeptide may comprise a hingeregion that has its origin in an immunoglobulin of a species, of animmunoglobulin isotype or class, or of an immunoglobulin subclass thatis different from that of the CH2 and CH3 domains. For instance, incertain embodiments of the invention, the binding domain-immunoglobulinfusion protein may comprise a binding domain polypeptide that is fusedto an immunoglobulin hinge region polypeptide comprising a wild-typehuman IgA hinge region polypeptide, or a mutated human IgA hinge regionpolypeptide that contains zero or only one cysteine residues, asdescribed herein, or a wild-type human IgG1 hinge region polypeptide ora wild-type human IgE hinge region polypeptide or a mutated human IgG1hinge region polypeptide that is mutated to contain zero, one or twocysteine residues wherein the first cysteine of the wild-type hingeregion is not mutated, as also described herein. Such a hinge regionpolypeptide may be fused to an immunoglobulin heavy chain CH2 regionpolypeptide from a different Ig isotype or class, for example an IgA oran IgE or an IgG subclass, which in certain preferred embodiments willbe the IgG1 subclass and in certain other preferred embodiments may beany one of the IgG2, IgG3 or IgG4 subclasses.

For example, and as described in greater detail below, in certainembodiments of the present invention an immunoglobulin hinge regionpolypeptide is selected which is derived from a wild-type human IgAhinge region that naturally comprises three cysteines, where theselected hinge region polypeptide is truncated relative to the completehinge region such that only one of the cysteine residues remains (e.g.,SEQ ID NOS:35-36). Similarly, in certain other embodiments of theinvention, the binding domain-immunoglobulin fusion protein comprises abinding domain polypeptide that is fused to an immunoglobulin hingeregion polypeptide comprising a mutated hinge region polypeptide inwhich the number of cysteine residues is reduced by amino acidsubstitution or deletion, for example a mutated IgG1 hinge regioncontaining zero, one or two cysteine residues as described herein. Amutated hinge region polypeptide may thus be derived from a wild-typeimmunoglobulin hinge region that contains one or more cysteine residues.In certain embodiments, a mutated hinge region polypeptide may containzero or only one cysteine residue, wherein the mutated hinge regionpolypeptide is derived from a wild type immunoglobulin hinge region thatcontains, respectively, one or more or two or more cysteine residues. Inthe mutated hinge region polypeptide, the cysteine residues of thewild-type immunoglobulin hinge region are preferably substituted withamino acids that are incapable of forming a disulfide bond. In oneembodiment of the invention, the mutated hinge region polypeptide isderived from a human IgG wild-type hinge region polypeptide, which mayinclude any of the four human IgG isotype subclasses, IgG1, IgG2, IgG3or IgG4. In certain preferred embodiments, the mutated hinge regionpolypeptide is derived from a human IgG1 wild-type hinge regionpolypeptide. By way of example, a mutated hinge region polypeptidederived from a human IgG1 wild-type hinge region polypeptide maycomprise mutations at two of the three cysteine residues in thewild-type immunoglobulin hinge region, or mutations at all threecysteine residues.

The cysteine residues that are present in a wild-type immunoglobulinhinge region and that are removed by mutagenesis according toparticularly preferred embodiments of the present invention includecysteine residues that form, or that are capable of forming, interchaindisulfide bonds. Without wishing to be bound by theory, the presentinvention contemplates that mutation of such hinge region cysteineresidues, which are believed to be involved in formation of interchaindisulfide bridges, reduces the ability of the subject invention bindingdomain-immunoglobulin fusion protein to dimerize (or form higheroligomers) via interchain disulfide bond formation, while surprisinglynot ablating the ability of the fusion protein to promote antibodydependent cell-mediated cytotoxicity (ADCC) or to fix complement. Inparticular, the Fc receptors (FcR) which mediate ADCC (e.g., FcRIII,CD16) exhibit low affinity for immunoglobulin Fc domains, suggestingthat functional binding of Fc to FcR requires avidity stabilization ofthe Fc-FcR complex by virtue of the dimeric structure of heavy chains ina conventional antibody, and/or FcR aggregation and cross-linking by aconventional Ab Fc structure. (Sonderman et al., 2000 Nature 406:267;Radaev et al., 2001 J. Biol. Chem. 276:16469; Radaev et al., 2001 J.Biol. Chem. 276:16478; Koolwijk et al., 1989 J. Immunol. 143:1656; Katoet al., 2000 Immunol. Today 21:310.) Hence, the bindingdomain-immunoglobulin fusion proteins of the present invention providethe advantages associated with single-chain immunoglobulin fusionproteins while also unexpectedly retaining immunological activity.Similarly, the ability to fix complement is typically associated withimmunoglobulins that are dimeric with respect to heavy chain constantregions such as those that comprise Fc, while the bindingdomain-immunoglobulin fusion proteins of the present invention, whichmay, due to the replacement or deletion of hinge region cysteineresidues or due to other structural modifications as described herein,have compromised or ablated abilities to form interchain disulfidebonds, exhibit the unexpected ability to fix complement. Additionally,according to certain embodiments of the present invention wherein abinding domain-immunoglobulin fusion protein may comprise one or more ofa human IgE hinge region polypeptide, a human IgE CH2 constant regionpolypeptide, a human IgE CH3 constant region polypeptide, and a humanIgE CH4 constant region polypeptide, the invention fusion proteinsunexpectedly retain the immunological activity of mediating ADCC and/orof inducing an allergic response mechanism.

Selection of an immunoglobulin hinge region polypeptide according tocertain embodiments of the subject invention bindingdomain-immunoglobulin fusion proteins may relate to the use of an“alternative hinge region” polypeptide sequence, which includes apolypeptide sequence that is not necessarily derived from anyimmunoglobulin hinge region sequence per se. Instead, an alternativehinge region refers to a hinge region polypeptide that comprises anamino acid sequence of at least ten consecutive amino acids, and incertain embodiments at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21-25, 26-30, 31-50, 51-60, 71-80, 81-90, or 91-110 amino acids that ispresent in a sequence selected from any one of SEQ ID NOS: 215, 216,217, 218, 223, 224, 6, 15, 16. 35, 36, 37, 41,207, 208, 223, 275, 276,277, 296, 300, 350, 390, 391, 392, 396, 397, 398, 488, 582, 584, 586,for example a polypeptide sequence derived from a region located betweenintrachain disulfide-generated immunoglobulin-like loop domains ofimmunoglobulin gene superfamily members such as CD2 (e.g., Genbank Acc.No. NM_(—)001767), CD4 (e.g., Genbank Acc. No. NM_(—)000616), CD5 (e.g.,Genbank Acc. No. BC027901), CD6 (e.g., Genbank Acc. No. NM_(—)006725),CD7 (e.g., Genbank Acc. Nos. XM_(—)046782, BC009293, NM_(—)006137) orCD8 (e.g., Genbank Acc. No. M12828), or other Ig superfamily members. Byway of non-limiting example, an alternative hinge region may provide aglycosylation site as provided herein, or may provide a humangene-derived polypeptide sequence for purposes of enhancing the degreeof “humanization” of a fusion protein, or may comprise an amino acidsequence that reduces the ability of a fusion protein to form multimersor oligomers or aggregates or the like. Certain alternative hinge regionpolypeptide sequences as described herein may be derived from thepolypeptide sequences of immunoglobulin gene superfamily members thatare not actual immunoglobulins per se. For instance and according tonon-limiting theory, certain polypeptide sequences that are situatedbetween intrachain disulfide-generated immunoglobulin loop domain ofimmunoglobulin gene super-family member proteins may be used asalternative hinge region polypeptides as provided herein, or may befurther modified for such use.

As noted above, binding domain-immunoglobulin fusion proteins arebelieved, according to non-limiting theory, to be compromised in theirability to dimerize via interchain disulfide bond formation, and furtheraccording to theory, this property is a consequence of a reduction inthe number of cysteine residues that are present in the immunoglobulinhinge region polypeptide selected for inclusion in the construction ofthe fusion protein. Determination of the relative ability of apolypeptide to dimerize is well within the knowledge of the relevantart, where any of a number of established methodologies may be appliedto detect protein dimerization (see, e.g., Scopes, Protein Purification:Principles and Practice, 1987 Springer-Verlag, New York). For example,biochemical separation techniques for resolving proteins on the basis ofmolecular size (e.g., gel electrophoresis, gel filtrationchromatography, analytical ultracentrifugation, etc.), and/or comparisonof protein physicochemical properties before and after introduction ofsulfhydryl-active (e.g., iodoacetamide, N-ethylmaleimide) ordisulfide-reducing (e.g., 2-mercaptoethanol, dithiothreitol) agents, orother equivalent methodologies, may all be employed for determining adegree of polypeptide dimerization or oligomerization, and fordetermining possible contribution of disulfide bonds to such potentialquarternary structure. In certain embodiments, the invention relates toa binding domain-immunoglobulin fusion protein that exhibits a reduced(i.e., in a statistically significant manner relative to an appropriateIgG-derived control) ability to dimerize, relative to a wild-type humanimmunoglobulin G hinge region polypeptide as provided herein.Accordingly, those familiar with the art will be able readily todetermine whether a particular fusion protein displays such reducedability to dimerize.

Compositions and methods for preparation of immunoglobulin fusionproteins are well known in the art, as described for example, in U.S.Pat. No. 5,892,019, which discloses recombinant antibodies that are theproducts of a single encoding polynucleotide but which are not bindingdomain-immunoglobulin fusion proteins according to the presentinvention.

For an immunoglobulin fusion protein of the invention which is intendedfor use in humans, the constant regions will typically be of humansequence origin, to minimize a potential anti-human immune response andto provide appropriate effector functions. Manipulation of sequencesencoding antibody constant regions is described in the PCT publicationof Morrison and Oi, WO 89/07142. In particularly preferred embodiments,the CH1 domain is deleted and the carboxyl end of the binding domain, orwhere the binding domain comprises two immunoglobulin variable regionpolypeptides, the second (i.e., more proximal to the C-terminus)variable region is joined to the amino terminus of CH2 through the hingeregion. A schematic diagram depicting the structures of two exemplarybinding domain-immunoglobulin fusion proteins is shown in FIG. 11, whereit should be noted that in particularly preferred embodiments nointerchain disulfide bonds are present, and in other embodiments arestricted number of interchain disulfide bonds may be present relativeto the number of such bonds that would be present if wild-type hingeregion polypeptides were instead present, and that in other embodimentsthe fusion protein comprises a mutated hinge region polypeptide thatexhibits a reduced ability to dimerize, relative to a wild-type humanIgG hinge region polypeptide. Thus, the isolated polynucleotide moleculecodes for a single chain immunoglobulin fusion protein having a bindingdomain that provides specific binding affinity for a selected antigen.

The invention also contemplates in certain embodiments bindingdomain-immunoglobulin fusion proteins as provided herein that comprisefused polypeptide sequences or portions thereof derived from a pluralityof genetic sources, for example, according to molecular “domainswapping” paradigms. Those having familiarity with the art will readilyappreciate that selection of such polypeptide sequences for assemblyinto a binding domain-immunoglobulin fusion protein may involvedetermination of what are appropriate portions of each componentpolypeptide sequence, based on structural and/or functional propertiesof each such sequence (see, e.g., Carayannopoulos et al., 1996 J. Exp.Med. 183:1579; Harlow et al., Eds., Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor (1988)). The componentpolypeptide sequences of which the fusion protein is comprised maytherefore comprise intact or full-length binding domain, immunoglobulin,linker and/or plasma membrane anchor domain polypeptide sequences, ortruncated versions or variants thereof as provided herein. According tothese and related embodiments of the invention, any two or more of thecandidate component polypeptides of which the subject invention fusionprotein may be comprised will be derived from independent sources, suchas from immunoglobulin sequences of differing allotype, isotype,subclass, class, or species of origin (e.g., xenotype). Thus, as anon-limiting example, a binding domain polypeptide (or its constituentpolypeptides such as one or more variable region polypeptides and/or alinker polypeptide), a hinge region polypeptide, immunoglobulin heavychain CH2 and CH3 constant region polypeptides and optionally animmunoglobulin heavy chain CH4 constant region polypeptide as may beobtained from an IgM or IgE heavy chain, and a plasma membrane anchordomain polypeptide may all be separately obtained from distinct geneticsources and engineered into a chimeric or fusion protein using wellknown techniques and according to methodologies described herein.

Accordingly, a binding domain-immunoglobulin fusion protein according tocertain embodiments of the present invention may also therefore comprisein pertinent part an immunoglobulin heavy chain CH3 constant regionpolypeptide that is a wild-type IgA CH3 constant region polypeptide, oralternatively, that is a mutated IgA CH3 constant region polypeptidethat is incapable of associating with a J chain; preferably the IgA CH3constant region polypeptides are of human origin. By way of briefbackground, IgA molecules are known to be released into secretory fluidsby a mechanism that involves association of IgA into disulfide-linkedpolymers (e.g., dimers) via a J chain polypeptide (e.g., Genbank Acc.Nos. XM_(—)059628, M12378, M12759; Johansen et al., 1999 Eur. J.Immunol. 29:1701) and interaction of the complex so formed with anotherprotein that acts as a receptor for polymeric immunoglobulin, and whichis known as transmembrane secretory component (S C; Johansen et al.,2000 Sc. J. Immunol. 52:240; see also, e.g., Sorensen et al., 2000 Int.Immunol. 12:19; Yoo et al., 1999 J. Biol. Chem. 274:33771; Yoo et al.,2002 J. Immunol. Meth. 261:1; Corthesy, 2002 Trends Biotechnol. 20:65;Symersky et al., 2000 Mol. Immunol. 37:133; Crottet et al., 1999Biochem. J. 341:299). Interchain disulfide bond formation between IgA Fcdomains and J chain is mediated through a penultimate cysteine residuein an 18-amino acid C-terminal extension that forms part of the IgAheavy chain constant region CH3 domain polypeptide (Yoo et al., 1999;Sorensen et al., 2000). Certain embodiments of the subject inventionfusion proteins therefore contemplate inclusion of the wild-type IgAheavy chain constant region polypeptide sequence, which is capable ofassociating with J chain. Certain other embodiments of the invention,however, contemplate fusion proteins that comprise a mutated IgA CH3constant region polypeptide that is incapable of associating with a Jchain. According to such embodiments, two or more residues from theC-terminus of an IgA CH3 constant region polypeptide such as a human IgACH3 constant region polypeptide may be deleted to yield a truncated CH3constant region polypeptide as provided herein. In preferred embodimentsand as described in greater detail below, a mutated human IgA CH3constant region polypeptide that is incapable of associating with a Jchain comprises such a C-terminal deletion of either four or 18 aminoacids. However, the invention need not be so limited, such that themutated IgA CH3 constant region polypeptide may comprise a deletion of2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21-25, 26-30 or more amino acids, so long as the fusion protein iscapable of specifically binding an antigen and of at least oneimmunological activity as provided herein. Alternatively, the inventionalso contemplates fusion proteins that comprise a mutated IgA CH3constant region polypeptide that is incapable of associating with a Jchain by virtue of replacement of the penultimate cysteine, or bychemical modification of that amino acid residue, in a manner thatprevents interchain disulfide bond formation. Methods for determiningwhether a fusion protein can associate with a J chain will be known tothose having familiarity with the art and are described herein,including in the cited references.

As also described herein and according to procedures known in the art,the fusion protein may further be tested routinely for immunologicalactivity, for instance, in ADCC or CDC assays. As an illustrativeexample, a fusion protein according to such an embodiment may comprise abinding domain polypeptide derived from a human heavy chain variableregion polypeptide sequence, a human IgA-derived immunoglobulin hingeregion polypeptide sequence, a human IgG1 immunoglobulin heavy chain CH2constant region polypeptide sequence, a human IgG2 immunoglobulin heavychain CH3 constant region polypeptide sequence, and optionally a humanIgE immunoglobulin heavy chain CH4 constant region polypeptide sequenceand/or a human TNF-α receptor type 1 (TNFR1) plasma membrane anchordomain polypeptide sequence that comprises a cytoplasmic tailpolypeptide which is capable of apoptotic signaling. The inventiontherefore contemplates these and other embodiments according to thepresent invention in which two or more polypeptide sequences that arepresent in a fusion protein have independent genetic origins.

As noted above, in certain embodiments the bindingprotein-immunoglobulin fusion protein comprises at least oneimmunoglobulin variable region polypeptide, which may be a light chainor a heavy chain variable region polypeptide, and in certain embodimentsthe fusion protein comprises at least one such light chain V-region andone such heavy chain V-region and at least one linker peptide that isfused to each of the V-regions. Construction of such binding domains,for example single chain Fv domains, is well known in the art and isdescribed in greater detail in the Examples below, and has beendescribed, for example, in U.S. Pat. No. 5,892,019 and references citedtherein; selection and assembly of single-chain variable regions and oflinker polypeptides that may be fused to each of a heavy chain-derivedand a light chain-derived V region (e.g., to generate a binding domainthat comprises a single-chain Fv polypeptide) is also known to the artand described herein and, for example, in U.S. Pat. No. 5,869,620, U.S.Pat. No. 4,704,692 and U.S. Pat. No. 4,946,778. In certain embodimentsall or a portion of an immunoglobulin sequence that is derived from anon-human source may be “humanized” according to recognized proceduresfor generating humanized antibodies, i.e., immunoglobulin sequences intowhich human Ig sequences are introduced to reduce the degree to which ahuman immune system would perceive such proteins as foreign (see, e.g.,U.S. Pat. Nos. 5,693,762; 5,585,089; 4,816,567; 5,225,539; 5,530,101;and references cited therein).

Binding domain-immunoglobulin fusion proteins as described herein may,according to certain embodiments, desirably comprise sites forglycosylation, e.g., covalent attachment of carbohydrate moieties suchas monosaccharides or oligosaccharides. Incorporation of amino acidsequences that provide substrates for polypeptide glycosylation iswithin the scope of the relevant art, including, for example, the use ofgenetic engineering or protein engineering methodologies to obtain apolypeptide sequence containing the classic Asn-X-Ser/Thr site forN-(asparagine)-linked glycosylation, or a sequence containing Ser or Thrresidues that are suitable substrates for O-linked glycosylation, orsequences amenable to C-mannosylation,glypiation/glycosylphosphatidylinositol modification, orphosphoglycation, all of which can be identified according toart-established criteria (e.g., Spiro, 2002 Glybiology 12:43R). Withoutwishing to be bound by theory, glycosylated fusion proteins havingparticular amino acid sequences may beneficially possess attributesassociated with one or more of improved solubility, enhanced stabilityin solution, enhanced physiological stability, improved bioavailabilityincluding in vivo biodistribution, and superior resistance to proteases,all in a statistically significant manner, relative to fusion proteinshaving the same or highly similar amino acid sequences but lackingglycosyl moieties. In certain preferred embodiments the subjectinvention fusion protein comprises a glycosylation site that is presentin a linker as provided herein, and in certain other preferredembodiments the subject invention fusion protein comprises aglycosylation site that is present in a hinge region polypeptidesequence as provided herein.

In certain preferred embodiments of the present invention, the bindingdomain-immunoglobulin fusion protein is a protein or glycoprotein thatis capable of being expressed by a host cell such that it localizes tothe cell surface. Binding domain-immunoglobulin fusion proteins thatlocalize to the cell surface may do so by virtue of having naturallypresent or artificially introduced structural features that direct thefusion protein to the cell surface (e.g., Nelson et al. 2001 Trends CellBiol. 11:483; Ammon et al., 2002 Arch. Physiol. Biochem. 110:137; Kasaiet al., 2001 J. Cell Sci. 114:3115; Watson et al., 2001 Am. J. Physiol.Cell Physiol. 281:C215; Chatterjee et al., 200 J. Biol. Chem. 275:24013)including by way of illustration and not limitation, secretory signalsequences, leader sequences, plasma membrane anchor domain polypeptidessuch as hydrophobic transmembrane domains (e.g., Heuck et al., 2002 CellBiochem. Biophys. 36:89; Sadlish et al., 2002 Biochem J. 364:777;Phoenix et al., 2002 Mol. Membr. Biol. 19:1; Minke et al., 2002 Physiol.Rev. 82:429) or glycosylphosphatidylinositol attachment sites(“glypiation” sites, e.g., Chatterjee et al., 2001 Cell Mol. Life Sci.58:1969; Hooper, 2001 Proteomics 1:748; Spiro, 2002 Glycobiol. 12:43R),cell surface receptor binding domains, extracellular matrix bindingdomains, or any other structural feature that causes the fusion proteinto localize to the cell surface. Particularly preferred are fusionproteins that comprise a plasma membrane anchor domain which includes atransmembrane polypeptide domain, typically comprising a membranespanning domain which includes a hydrophobic region capable ofenergetically favorable interaction with the phospholipid fatty acyltails that form the interior of the plasma membrane bilayer. Suchfeatures are well known to those of ordinary skill in the art, who willfurther be familiar with methods for introducing nucleic acid sequencesencoding these features into the subject expression constructs bygenetic engineering, and with routine testing of such constructs toverify cell surface localization of the product.

According to certain further embodiments, a plasma membrane anchordomain polypeptide comprises such a transmembrane domain polypeptide andalso comprises a cytoplasmic tail polypeptide, which refers to a regionor portion of the polypeptide sequence that contacts the cytoplasmicface of the plasma membrane and/or is in contact with the cytosol orother cytoplasmic components. A large number of cytoplasmic tailpolypeptides are known that comprise the intracellular portions ofplasma membrane transmembrane proteins, and discrete functions have beenidentified for many such polypeptides, including biological signaltransduction (e.g., activation or inhibition of protein kinases, proteinphosphatases, G-proteins, cyclic nucleotides and other secondmessengers, ion channels, secretory pathways), biologically activemediator release, stable or dynamic association with one or morecytoskeletal components, cellular differentiation, cellular activation,mitogenesis, cytostasis, apoptosis and the like (e.g., Maher et al.,2002 Immunol. Cell Biol. 80:131; El Far et al., 2002 Biochem J. 365:329;Teng et al., 2002 Genome Biol. 2REVIEWS:3012; Simons et al., 2001 CellSignal 13:855; Furie et al., 2001 Thromb. Haemost. 86:214; Gaffen, 2001Cytokine 14:63; Dittel, 2000 Arch. Immunol. Ther. Exp. (Warsz.) 48:381;Parnes et al., 2000 Immunol. Rev. 176:75; Moretta et al., 2000 Semin.Immunol. 12:129; Ben Ze'ev, 1999 Ann. N.Y. Acad. Sci. 886:37; Marsterset al., Recent Prog. Horm. Res. 54:225).

In the context of methods of using binding domain-immunoglobulin fusionproteins for the treatment of a malignant condition or a B-cell disorderas provided herein, the present invention contemplates certainembodiments wherein a binding domain-immunoglobulin fusion protein thatcomprises a plasma membrane anchor domain polypeptide is expressed at acell surface and further comprises a cytoplasmic tail polypeptide whichcomprises an apoptosis signaling polypeptide sequence. A number ofapoptosis signaling polypeptide sequences are known to the art, asreviewed, for example, in When Cells Die: A Comprehensive Evaluation ofApoptosis and Programmed Cell Death (R. A. Lockshin et al., Eds., 1998John Wiley & Sons, New York; see also, e.g., Green et al., 1998 Science281:1309 and references cited therein; Ferreira et al., 2002 Clin. Canc.Res. 8:2024; Gurumurthy et al., 2001 Cancer Metastas. Rev. 20:225;Kanduc et al., 2002 Int. J. Oncol. 21:165). Typically an apoptosissignaling polypeptide sequence comprises all or a portion of, or isderived from, a receptor death domain polypeptide, for instance, FADD(e.g., Genbank Acc. Nos. U24231, U43184, AF009616, AF009617,NM_(—)012115), TRADD (e.g., Genbank Acc. No. NM_(—)003789), RAIDD (e.g.,Genbank Acc. No. U87229), CD95 (FAS/Apo-1; e.g., Genbank Acc. Nos.X89101, NM_(—)003824, AF344850, AF344856), TNF-α-receptor-1 (TNFR1,e.g., Genbank Acc. Nos. 563368, AF040257), DR5 (e.g., Genbank Acc. No.AF020501, AF016268, AF012535), an ITIM domain (e.g., Genbank Acc. Nos.AF081675, BC015731, NM_(—)006840, NM_(—)006844, NM_(—)006847,XM_(—)017977; see, e.g., Billadeau et al., 2002 J. Clin. Invest.109:161), an ITAM domain (e.g., Genbank Acc. Nos. NM_(—)005843,NM_(—)003473, BC030586; see, e.g., Billadeau et al., 2002), or otherapoptosis-associated receptor death domain polypeptides known to theart, for example, TNFR2 (e.g., Genbank Acc. No. L49431, L49432),caspase/procaspase-3 (e.g., Genbank Acc. No. XM_(—)54686),caspase/procaspase-8 (e.g., AF380342, NM_(—)004208, NM_(—)001228,NM_(—)033355, NM_(—)033356, NM_(—)033357, NM_(—)033358),caspase/procaspase-2 (e.g., Genbank Acc. No. AF314174, AF314175), etc.

Cells in a biological sample that are suspected of undergoing apoptosismay be examined for morphological, permeability or other changes thatare indicative of an apoptotic state. For example by way of illustrationand not limitation, apoptosis in many cell types may cause alteredmorphological appearance such as plasma membrane blebbing, cell shapechange, loss of substrate adhesion properties or other morphologicalchanges that can be readily detected by a person having ordinary skillin the art, for example by using light microscopy. As another example,cells undergoing apoptosis may exhibit fragmentation and disintegrationof chromosomes, which may be apparent by microscopy and/or through theuse of DNA-specific or chromatin-specific dyes that are known in theart, including fluorescent dyes. Such cells may also exhibit alteredplasma membrane permeability properties as may be readily detectedthrough the use of vital dyes (e.g., propidium iodide, trypan blue) orby the detection of lactate dehydrogenase leakage into the extracellularmilieu. These and other means for detecting apoptotic cells bymorphologic criteria, altered plasma membrane permeability and relatedchanges will be apparent to those familiar with the art.

In another embodiment of the invention wherein a bindingdomain-immunoglobulin fusion protein that is expressed at a cell surfacecomprises a plasma membrane anchor domain having a transmembrane domainand a cytoplasmic tail that comprises an apoptosis signalingpolypeptide, cells in a biological sample may be assayed fortranslocation of cell membrane phosphatidylserine (PS) from the inner tothe outer leaflet of the plasma membrane, which may be detected, forexample, by measuring outer leaflet binding by the PS-specific proteinannexin. (Martin et al., J. Exp. Med. 182:1545, 1995; Fadok et al., J.Immunol. 148:2207, 1992.) In still other related embodiments of theinvention, including embodiments wherein the bindingdomain-immunoglobulin fusion protein is expressed at the cell surfaceand comprises a plasma membrane anchor domain having an apoptosissignaling polypeptide and also including embodiments wherein the bindingdomain-immunoglobulin fusion protein is a soluble protein that lacks amembrane anchor domain and that is capable of inducing apoptosis, acellular response to an apoptogen is determined by an assay forinduction of specific protease activity in any member of a family ofapoptosis-activated proteases known as the caspases (see, e.g., Green etal., 1998 Science 281:1309). Those having ordinary skill in the art willbe readily familiar with methods for determining caspase activity, forexample by determination of caspase-mediated cleavage of specificallyrecognized protein substrates. These substrates may include, forexample, poly-(ADP-ribose) polymerase (PARP) or other naturallyoccurring or synthetic peptides and proteins cleaved by caspases thatare known in the art (see, e.g., Ellerby et al., 1997 J. Neurosci.17:6165). The synthetic peptide Z-Tyr-Val-Ala-Asp-AFC (SEQ IDNO:______;), wherein “Z” indicates a benzoyl carbonyl moiety and AFCindicates 7-amino-4-trifluoromethylcoumarin (Kluck et al., 1997 Science275:1132; Nicholson et al., 1995 Nature 376:37), is one such substrate.Other non-limiting examples of substrates include nuclear proteins suchas U1-70 kDa and DNA-PKcs (Rosen and Casciola-Rosen, 1997 J. Cell.Biochem. 64:50; Cohen, 1997 Biochem. J. 326:1). Cellular apoptosis mayalso be detected by determination of cytochrome c that has escaped frommitochondria in apoptotic cells (e.g., Liu et al., Cell 86:147, 1996).Such detection of cytochrome c may be performed spectrophotometrically,immunochemically or by other well established methods for determiningthe presence of a specific protein. Persons having ordinary skill in theart will readily appreciate that there may be other suitable techniquesfor quantifying apoptosis.

Once a binding domain-immunoglobulin fusion protein as provided hereinhas been designed, DNAs encoding the polypeptide may be synthesized viaoligonucleotide synthesis as described, for example, in Sinha et al.,Nucleic Acids Res., 12, 4539-4557 (1984); assembled via PCR asdescribed, for example in Innis, Ed., PCR Protocols, Academic Press(1990) and also in Better et al. J. Biol. Chem. 267, 16712-16118 (1992);cloned and expressed via standard procedures as described, for example,in Ausubel et al., Eds., Current Protocols in Molecular Biology, JohnWiley & Sons, New York (1989) and also in Robinson et al., Hum. Antibod.Hybridomas, 2, 84-93 (1991); and tested for specific antigen bindingactivity, as described, for example, in Harlow et al., Eds., Antibodies:A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, ColdSpring Harbor (1988) and Munson et al., Anal. Biochem., 107, 220-239(1980).

The preparation of single polypeptide chain binding molecules of the Fvregion, single-chain Fv molecules, is described in U.S. Pat. No.4,946,778, which is incorporated herein by reference. In the presentinvention, single-chain Fv-like molecules are synthesized by encoding afirst variable region of the heavy or light chain, followed by one ormore linkers to the variable region of the corresponding light or heavychain, respectively. The selection of appropriate linker(s) between thetwo variable regions is described in U.S. Pat. No. 4,946,778 (see also,e.g., Huston et al., 1993 Int. Rev. Immunol. 10:195). An exemplarylinker described herein is (Gly-Gly-Gly-Gly-Ser)₃. The linker is used toconvert the naturally aggregated but chemically separate heavy and lightchains into the amino terminal antigen binding portion of a singlepolypeptide chain, wherein this antigen binding portion will fold into astructure similar to the original structure made of two polypeptidechains and thus retain the ability to bind to the antigen of interest.The nucleotide sequences encoding the variable regions of the heavy andlight chains, joined by a sequence encoding a linker, are joined to anucleotide sequence encoding antibody constant regions. The constantregions are those which permit the resulting polypeptide to forminterchain disulfide bonds to form a dimer, and which contain desiredeffector functions, such as the ability to mediate antibody-dependentcellular cytotoxicity (ADCC). For an immunoglobulin-like molecule of theinvention which is intended for use in humans, the constant regions willtypically be substantially human to minimize a potential anti-humanimmune response and to provide appropriate effector functions.Manipulation of sequences encoding antibody constant regions isdescribed in the PCT publication of Morrison and Oi, WO 89/07142, whichis incorporated herein by reference. In preferred embodiments, the CH1domain is deleted and the carboxyl end of the binding domain polypeptide(e.g., an immunoglobulin variable region polypeptide) is joined to theamino terminus of CH2 via a hinge region polypeptide as provided herein.

As described above, the present invention provides recombinantexpression constructs capable of directing the expression of bindingdomain-immunoglobulin fusion proteins as provided herein. The aminoacids, which occur in the various amino acid sequences referred toherein, are identified according to their well known three-letter orsingle-letter abbreviations. The nucleotides, which occur in the variousDNA sequences or fragments thereof referred herein, are designated withthe standard single letter designations used routinely in the art. Agiven amino acid sequence may also encompass similar amino acidsequences having only minor changes, for example by way of illustrationand not limitation, covalent chemical modifications, insertions,deletions and substitutions, which may further include conservativesubstitutions. Amino acid sequences that are similar to one another mayshare substantial regions of sequence homology. In like fashion,nucleotide sequences may encompass substantially similar nucleotidesequences having only minor changes, for example by way of illustrationand not limitation, covalent chemical modifications, insertions,deletions and substitutions, which may further include silent mutationsowing to degeneracy of the genetic code. Nucleotide sequences that aresimilar to one another may share substantial regions of sequencehomology.

The presence of a malignant condition in a subject refers to thepresence of dysplastic, cancerous and/or transformed cells in thesubject, including, for example neoplastic, tumor, non-contact inhibitedor oncogenically transformed cells, or the like (e.g., melanoma,carcinomas such as adenocarcinoma, squamous cell carcinoma, small cellcarcinoma, oat cell carcinoma, etc., sarcomas such as chondrosarcoma,osteosarcoma, etc.) which are known to the art and for which criteriafor diagnosis and classification are established. In preferredembodiments contemplated by the present invention, for example, suchcancer cells are malignant hematopoietic cells, such as transformedcells of lymphoid lineage and in particular, B-cell lymphomas and thelike; cancer cells may in certain preferred embodiments also beepithelial cells such as carcinoma cells. The invention alsocontemplates B-cell disorders, which may include certain malignantconditions that affect B-cells (e.g., B-cell lymphoma) but which is notintended to be so limited, and which is also intended to encompassautoimmune diseases and in particular, diseases, disorders andconditions that are characterized by autoantibody production.

Autoantibodies are antibodies that react with self antigens.Autoantibodies are detected in several autoimmune diseases (i.e., adisease, disorder or condition wherein a host immune system generates aninappropriate anti-“self” immune reaction) where they are involved indisease activity. The current treatments for these autoimmune diseasesare immunosuppressive drugs that require continuing administration, lackspecificity, and cause significant side effects. New approaches that caneliminate autoantibody production with minimal toxicity will address anunmet medical need for a spectrum of diseases that affect many people.The subject invention binding domain-immunoglobulin fusion protein isdesigned for improved penetration into lymphoid tissues. Depletion of Blymphocytes interrupts the autoantibody production cycle, and allows theimmune system to reset as new B lymphocytes are produced from precursorsin the bone marrow.

A number of diseases have been identified for which beneficial effectsare believed, according to non-limiting theory, to result from B celldepletion therapy; a brief description of several exemplars of thesediseases follows.

Autoimmune thyroid disease includes Graves' disease and Hashimoto'sthyroiditis. In the United States alone, there are about 20 millionpeople who have some form of autoimmune thyroid disease. Autoimmunethyroid disease results from the production of autoantibodies thateither stimulate the thyroid to cause hyperthyroidism (Graves' disease)or destroy the thyroid to cause hypothyroidism (Hashimoto'sthyroiditis). Stimulation of the thyroid is caused by autoantibodiesthat bind and activate the thyroid stimulating hormone (TSH) receptor.Destruction of the thyroid is caused by autoantibodies that react withother thyroid antigens.

Current therapy for Graves' disease includes surgery, radioactiveiodine, or antithyroid drug therapy. Radioactive iodine is widely used,since antithyroid medications have significant side effects and diseaserecurrence is high. Surgery is reserved for patients with large goitersor where there is a need for very rapid normalization of thyroidfunction. There are no therapies that target the production ofautoantibodies responsible for stimulating the TSH receptor. Currenttherapy for Hashimoto's thyroiditis is levothyroxine sodium, and therapyis usually lifelong because of the low likelihood of remission.Suppressive therapy has been shown to shrink goiters in Hashimoto'sthyroiditis, but no therapies that reduce autoantibody production totarget the disease mechanism are known.

Rheumatoid arthritis (RA) is a chronic disease characterized byinflammation of the joints, leading to swelling, pain, and loss offunction. RA effects an estimated 2.5 million people in the UnitedStates. RA is caused by a combination of events including an initialinfection or injury, an abnormal immune response, and genetic factors.While autoreactive T cells and B cells are present in RA, the detectionof high levels of antibodies that collect in the joints, calledrheumatoid factor, is used in the diagnosis of RA. Current therapy forRA includes many medications for managing pain and slowing theprogression of the disease. No therapy has been found that can cure thedisease. Medications include nonsteroidal antiinflammatory drugs(NSAIDS), and disease modifying antirheumatic drugs (DMARDS). NSAIDS areeffective in benign disease, but fail to prevent the progression tojoint destruction and debility in severe RA. Both NSAIDS and DMARDS areassociated with significant side effects. Only one new DMARD,Leflunomide, has been approved in over 10 years. Leflunomide blocksproduction of autoantibodies, reduces inflammation, and slowsprogression of RA. However, this drug also causes severe side effectsincluding nausea, diarrhea, hair loss, rash, and liver injury.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease caused byrecurrent injuries to blood vessels in multiple organs, including thekidney, skin, and joints. SLE effects over 500,000 people in the UnitedStates. In patients with SLE, a faulty interaction between T cells and Bcells results in the production of autoantibodies that attack the cellnucleus. These include anti-double stranded DNA and anti-Sm antibodies.Autoantibodies that bind phospholipids are also found in about half ofSLE patients, and are responsible for blood vessel damage and low bloodcounts. Immune complexes accumulate the kidneys, blood vessels, andjoints of SLE patients, where they cause inflammation and tissue damage.No treatment for SLE has been found to cure the disease. NSAIDS andDMARDS are used for therapy depending upon the severity of the disease.Plasmapheresis with plasma exchange to remove autoantibodies can causetemporary improvement in SLE patients. There is general agreement thatautoantibodies are responsible for SLE, so new therapies that depletethe B cell lineage, allowing the immune system to reset as new B cellsare generated from precursors, offer hope for long lasting benefit inSLE patients.

Sjogren's syndrome is an autoimmune disease characterized by destructionof the body's moisture producing glands. Sjogren's syndrome is one ofthe most prevalent autoimmune disorders, striking up to 4 million peoplein the United States. About half of people with Sjogren's also have aconnective tissue disease, such as rheumatoid arthritis, while the otherhalf have primary Sjogren's with no other concurrent autoimmune disease.Autoantibodies, including anti-nuclear antibodies, rheumatoid factor,anti-fodrin, and anti-muscarinic receptor are often present in patientswith Sjogren's syndrome. Conventional therapy includes corticosteroids.

Immune Thrombocytopenic purpura (ITP) is caused by autoantibodies thatbind to blood platelets and cause their destruction. Some cases of ITPare caused by drugs, and others are associated with infection,pregnancy, or autoimmune disease such as SLE. About half of all casesare classified as “idiopathic”, meaning the cause is unknown. Thetreatment of ITP is determined by the severity of the symptoms. In somecases, no therapy is needed. In most cases, immunosuppressive drugs areused, including corticosteroids or intravenous infusions of immuneglobulin to deplete T cells. Another treatment that usually results inan increased number of platelets is removal of the spleen, the organthat destroys antibody-coated platelets. More potent immunosuppressivedrugs, including cyclosporine, cyclophosphamide, or azathioprine, areused for patients with severe cases. Removal of autoantibodies bypassage of patients' plasma over a Protein A column is used as a secondline treatment in patients with severe disease.

Multiple Sclerosis (MS) is an autoimmune disease characterized byinflammation of the central nervous system and destruction of myelin,which insulates nerve cell fibers in the brain, spinal cord, and body.Although the cause of MS is unknown, it is widely believed thatautoimmune T cells are primary contributors to the pathogenesis of thedisease. However, high levels of antibodies are present in the cerebralspinal fluid of patients with MS, and some theories predict that the Bcell response leading to antibody production is important for mediatingthe disease. No B cell depletion therapies have been studies in patientswith MS. There is no cure for MS. Current therapy is corticosteroids,which can reduce the duration and severity of attacks, but do not affectthe course of MS over time. New biotechnology interferon (IFN) therapiesfor MS have recently been approved.

Myasthenia Gravis (MG) is a chronic autoimmune neuromuscular disorderthat is characterized by weakness of the voluntary muscle groups. MGeffects about 40,000 people in the United States. MG is caused byautoantibodies that bind to acetylcholine receptors expressed atneuromuscular junctions. The autoantibodies reduce or blockacetylcholine receptors, preventing the transmission of signals fromnerves to muscles. There is no known cure for MG. Common treatmentsinclude immunosuppression with corticosteroids, cyclosporine,cyclophosphamide, or azathioprine. Surgical removal of the thymus isoften used to blunt the autoimmune response. Plasmapheresis, used toreduce autoantibody levels in the blood, is effective in MG, but isshort-lived because the production of autoantibodies continues.Plasmapheresis is usually reserved for severe muscle weakness prior tosurgery.

Psoriasis affects approximately five million people. It is an autoimmuneinflammation of the skin and joints. Psoriasis is associated witharthritis (psoriatic arthritis) in up to 30% of patients with psoriasis.Many treatments are used, including steroids, UV light retenoids,vitamin D derivatives, cyclosporine, and methotrexate.

Scleroderma is a chronic autoimmune disease of the connective tissuethat is also known as systemic sclerosis. Scleroderma is characterizedby an overproduction of collagen, resulting in a thickening of the skin.Approximately 300,000 people in the United States have scleroderma.

Inflammatory Bowel Disease, including Crohn's disease and ulcerativecolitis, is an autoimmune disease of the digestive system.

The present invention further relates to constructs encoding bindingdomain-immunoglobulin fusion proteins, and in particular to methods foradministering recombinant constructs encoding such proteins that may beexpressed, for example, as fragments, analogs and derivatives of suchpolypeptides. The terms “fragment,” “derivative” and “analog” whenreferring to binding domain-immunoglobulin fusion polypeptides or fusionproteins, refers to any binding domain-immunoglobulin fusion polypeptideor fusion protein that retains essentially the same biological functionor activity as such polypeptide. Thus, an analog includes a proproteinwhich can be activated by cleavage of the proprotein portion to producean active binding domain-immunoglobulin fusion polypeptide.

A fragment, derivative or analog of an binding domain-immunoglobulinfusion polypeptide or fusion protein, including bindingdomain-immunoglobulin fusion polypeptides or fusion proteins encoded bythe cDNAs referred to herein, may be (i) one in which one or more of theamino acid residues are substituted with a conserved or non-conservedamino acid residue (preferably a conserved amino acid residue) and suchsubstituted amino acid residue may or may not be one encoded by thegenetic code, or (ii) one in which one or more of the amino acidresidues includes a substituent group, or (iii) one in which additionalamino acids are fused to the binding domain-immunoglobulin fusionpolypeptide, including amino acids that are employed for detection orspecific functional alteration of the binding domain-immunoglobulinfusion polypeptide or a proprotein sequence. Such fragments, derivativesand analogs are deemed to be within the scope of those skilled in theart from the teachings herein.

The polypeptides of the present invention include bindingdomain-immunoglobulin fusion polypeptides and fusion proteins havingbinding domain polypeptide amino acid sequences that are identical orsimilar to sequences known in the art, or fragments or portions thereof.For example by way of illustration and not limitation, a human CD154molecule extracellular domain [SEQ ID NO:_] is contemplated for useaccording to the instant invention, as are polypeptides having at least70% similarity (preferably a 70% identity) and more preferably 90%similarity (more preferably a 90% identity) to the reported polypeptideand still more preferably a 95% similarity (still more preferably a 95%identity) to the reported polypeptides and to portions of suchpolypeptides, wherein such portions of a binding domain-immunoglobulinfusion polypeptide generally contain at least 30 amino acids and morepreferably at least 50 amino acids. Extracellular domains includeportions of a cell surface molecule, and in particularly preferredembodiments cell surface molecules that are integral membrane proteinsor that comprise a plasma membrane spanning transmembrane domain, thatextend beyond the outer leaflet of the plasma membrane phospholipidbilayer when the molecule is expressed at a cell surface, preferably ina manner that exposes the extracellular domain portion of such amolecule to the external environment of the cell, also known as theextracellular milieu. Methods for determining whether a portion of acell surface molecule comprises an extracellular domain are well knownto the art and include experimental determination (e.g., direct orindirect labeling of the molecule, evaluation of whether the moleculecan be structurally altered by agents to which the plasma membrane isnot permeable such as proteolytic or lipolytic enzymes) or topologicalprediction based on the structure of the molecule (e.g., analysis of theamino acid sequence of a polypeptide) or other methodologies.

As known in the art, “similarity” between two polypeptides is determinedby comparing the amino acid sequence and conserved amino acidsubstitutes thereto of the polypeptide to the sequence of a secondpolypeptide. Fragments or portions of the nucleic acids encodingpolypeptides of the present invention may be used to synthesizefull-length nucleic acids of the present invention. As used herein, “%identity” refers to the percentage of identical amino acids situated atcorresponding amino acid residue positions when two or more polypeptideare aligned and their sequences analyzed using a gapped BLAST algorithm(e.g., Altschul et al., 1997 Nucl. Ac. Res. 25:3389) which weightssequence gaps and sequence mismatches according to the defaultweightings provided by the National Institutes of Health/NCBI database(Bethesda, Md.; see www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-newblast).

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally occurring nucleic acid orpolypeptide present in a living animal is not isolated, but the samenucleic acid or polypeptide, separated from some or all of theco-existing materials in the natural system, is isolated. Such nucleicacids could be part of a vector and/or such nucleic acids orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region “leader and trailer” as well as intervening sequences(introns) between individual coding segments (exons).

As described herein, the invention provides bindingdomain-immunoglobulin fusion proteins encoded by nucleic acids that havethe binding domain coding sequence fused in frame to an additionalimmunoglobulin domain encoding sequence to provide for expression of abinding domain polypeptide sequence fused to an additional functionalpolypeptide sequence that permits, for example by way of illustrationand not limitation, detection, functional alteration, isolation and/orpurification of the fusion protein. Such fusion proteins may permitfunctional alteration of a binding domain by containing additionalimmunoglobulin-derived polypeptide sequences that influence behavior ofthe fusion product, for example (and as described above) by reducing theavailability of sulfhydryl groups for participation in disulfide bondformation, and by conferring the ability to potentiate ADCC and/or CDC.

Modification of the polypeptide may be effected by any means known tothose of skill in this art. The preferred methods herein rely onmodification of DNA encoding the fusion protein and expression of themodified DNA. DNA encoding one of the binding domain-immunoglobulinfusions discussed above may be mutagenized using standard methodologies,including those described below. For example, cysteine residues that mayotherwise facilitate multimer formation or promote particular molecularconformations can be deleted from a polypeptide or replaced, e.g.,cysteine residues that are responsible for aggregate formation. Ifnecessary, the identity of cysteine residues that contribute toaggregate formation may be determined empirically, by deleting and/orreplacing a cysteine residue and ascertaining whether the resultingprotein aggregates in solutions containing physiologically acceptablebuffers and salts. In addition, fragments of bindingdomain-immunoglobulin fusions may be constructed and used. As notedabove, the counterreceptor/ligand binding domains for many candidatebinding domain-immunoglobulin fusion proteins have been delineated, suchthat one having ordinary skill in the art may readily select appropriatepolypeptide domains for inclusion in the encoded products of the instantexpression constructs.

Conservative substitutions of amino acids are well-known and may be madegenerally without altering the biological activity of the resultingbinding domain-immunoglobulin fusion protein molecule. For example, suchsubstitutions are generally made by interchanging within the groups ofpolar residues, charged residues, hydrophobic residues, small residues,and the like. If necessary, such substitutions may be determinedempirically merely by testing the resulting modified protein for theability to bind to the appropriate cell surface receptors in in vitrobiological assays, or to bind to appropriate antigens or desired targetmolecules.

The present invention further relates to nucleic acids which hybridizeto binding domain-immunoglobulin fusion protein encoding polynucleotidesequences as provided herein, or their complements, as will be readilyapparent to those familiar with the art, if there is at least 70%,preferably 80-85%, more preferably at least 90%, and still morepreferably at least 95%, 96%, 97%, 98% or 99% identity between thesequences. The present invention particularly relates to nucleic acidswhich hybridize under stringent conditions to the bindingdomain-immunoglobulin fusion encoding nucleic acids referred to herein.As used herein, the term “stringent conditions” means hybridization willoccur only if there is at least 90-95% and preferably at least 97%identity between the sequences. The nucleic acids which hybridize tobinding domain-immunoglobulin fusion encoding nucleic acids referred toherein, in preferred embodiments, encode polypeptides which retainsubstantially the same biological function or activity as the bindingdomain-immunoglobulin fusion polypeptides encoded by the cDNAs of thereferences cited herein.

As used herein, to “hybridize” under conditions of a specifiedstringency is used to describe the stability of hybrids formed betweentwo single-stranded nucleic acid molecules. Stringency of hybridizationis typically expressed in conditions of ionic strength and temperatureat which such hybrids are annealed and washed. Typically “high”,“medium” and “low” stringency encompass the following conditions orequivalent conditions thereto: high stringency: 0.1×SSPE or SSC, 0.1%SDS, 65° C.; medium stringency: 0.2×SSPE or SSC, 0.1% SDS, 50° C.; andlow stringency: 1.0×SSPE or SSC, 0.1% SDS, 50° C. As known to thosehaving ordinary skill in the art, variations in stringency ofhybridization conditions may be achieved by altering the time,temperature and/or concentration of the solutions used forprehybridization, hybridization and wash steps, and suitable conditionsmay also depend in part on the particular nucleotide sequences of theprobe used, and of the blotted, proband nucleic acid sample.Accordingly, it will be appreciated that suitably stringent conditionscan be readily selected without undue experimentation where a desiredselectivity of the probe is identified, based on its ability tohybridize to one or more certain proband sequences while not hybridizingto certain other proband sequences.

The nucleic acids of the present invention, also referred to herein aspolynucleotides, may be in the form of RNA or in the form of DNA, whichDNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may bedouble-stranded or single-stranded, and if single stranded may be thecoding strand or non-coding (anti-sense) strand. A coding sequence whichencodes an binding domain-immunoglobulin fusion polypeptide for useaccording to the invention may be identical to the coding sequence knownin the art for any given binding domain-immunoglobulin fusion, or may bea different coding sequence, which, as a result of the redundancy ordegeneracy of the genetic code, encodes the same bindingdomain-immunoglobulin fusion polypeptide.

The nucleic acids which encode binding domain-immunoglobulin fusionpolypeptides for use according to the invention may include, but are notlimited to: only the coding sequence for the bindingdomain-immunoglobulin fusion polypeptide; the coding sequence for thebinding domain-immunoglobulin fusion polypeptide and additional codingsequence; the coding sequence for the binding domain-immunoglobulinfusion polypeptide (and optionally additional coding sequence) andnon-coding sequence, such as introns or non-coding sequences 5′ and/or3′ of the coding sequence for the binding domain-immunoglobulin fusionpolypeptide, which for example may further include but need not belimited to one or more regulatory nucleic acid sequences that may be aregulated or regulatable promoter, enhancer, other transcriptionregulatory sequence, repressor binding sequence, translation regulatorysequence or any other regulatory nucleic acid sequence. Thus, the term“nucleic acid encoding” or “polynucleotide encoding” a bindingdomain-immunoglobulin fusion protein encompasses a nucleic acid whichincludes only coding sequence for a binding domain-immunoglobulin fusionpolypeptide as well as a nucleic acid which includes additional codingand/or non-coding sequence(s).

Nucleic acids and oligonucleotides for use as described herein can besynthesized by any method known to those of skill in this art (see,e.g., WO 93/01286, U.S. application Ser. No. 07/723,454; U.S. Pat. No.5,218,088; U.S. Pat. No. 5,175,269; U.S. Pat. No. 5,109,124).Identification of oligonucleotides and nucleic acid sequences for use inthe present invention involves methods well known in the art. Forexample, the desirable properties, lengths and other characteristics ofuseful oligonucleotides are well known. In certain embodiments,synthetic oligonucleotides and nucleic acid sequences may be designedthat resist degradation by endogenous host cell nucleolytic enzymes bycontaining such linkages as: phosphorothioate, methylphosphonate,sulfone, sulfate, ketyl, phosphorodithioate, phosphoramidate, phosphateesters, and other such linkages that have proven useful in antisenseapplications (see, e.g., Agrwal et al., Tetrahedron Lett. 28:3539-3542(1987); Miller et al., J. Am. Chem. Soc. 93:6657-6665 (1971); Stec etal., Tetrahedron Lett. 26:2191-2194 (1985); Moody et al., Nucl. AcidsRes. 12:4769-4782 (1989); Uznanski et al., Nucl. Acids Res. (1989);Letsinger et al., Tetrahedron 40:137-143 (1984); Eckstein, Annu. Rev.Biochem. 54:367-402 (1985); Eckstein, Trends Biol. Sci. 14:97-100(1989); Stein In: Oligodeoxynucleotides. Antisense Inhibitors of GeneExpression, Cohen, Ed., Macmillan Press, London, pp. 97-117 (1989);Jager et al., Biochemistry 27:7237-7246 (1988)).

In one embodiment, the present invention provides truncated components(e.g., binding domain polypeptide, hinge region polypeptide, linker,etc.) for use in a binding domain-immunoglobulin fusion protein, and inanother embodiment the invention provides nucleic acids encoding abinding domain-immunoglobulin fusion protein having such truncatedcomponents. A truncated molecule may be any molecule that comprises lessthan a full length version of the molecule. Truncated molecules providedby the present invention may include truncated biological polymers, andin preferred embodiments of the invention such truncated molecules maybe truncated nucleic acid molecules or truncated polypeptides. Truncatednucleic acid molecules have less than the full length nucleotidesequence of a known or described nucleic acid molecule, where such aknown or described nucleic acid molecule may be a naturally occurring, asynthetic or a recombinant nucleic acid molecule, so long as one skilledin the art would regard it as a full length molecule. Thus, for example,truncated nucleic acid molecules that correspond to a gene sequencecontain less than the full length gene where the gene comprises codingand non-coding sequences, promoters, enhancers and other regulatorysequences, flanking sequences and the like, and other functional andnon-functional sequences that are recognized as part of the gene. Inanother example, truncated nucleic acid molecules that correspond to amRNA sequence contain less than the full length mRNA transcript, whichmay include various translated and non-translated regions as well asother functional and non-functional sequences.

In other preferred embodiments, truncated molecules are polypeptidesthat comprise less than the full length amino acid sequence of aparticular protein or polypeptide component. As used herein “deletion”has its common meaning as understood by those familiar with the art, andmay refer to molecules that lack one or more of a portion of a sequencefrom either terminus or from a non-terminal region, relative to acorresponding full length molecule, for example, as in the case oftruncated molecules provided herein. Truncated molecules that are linearbiological polymers such as nucleic acid molecules or polypeptides mayhave one or more of a deletion from either terminus of the molecule or adeletion from a non-terminal region of the molecule, where suchdeletions may be deletions of 1-1500 contiguous nucleotide or amino acidresidues, preferably 1-500 contiguous nucleotide or amino acid residuesand more preferably 1-300 contiguous nucleotide or amino acid residues,including deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31-40,41-50, 51-74, 75-100, 101-150, 151-200, 201-250 or 251-299 contiguousnucleotide or amino acid residues. In certain particularly preferredembodiments truncated nucleic acid molecules may have a deletion of270-330 contiguous nucleotides. In certain other particularly preferredembodiments truncated polypeptide molecules may have a deletion of80-140 contiguous amino acids.

The present invention further relates to variants of the hereinreferenced nucleic acids which encode fragments, analogs and/orderivatives of a binding domain-immunoglobulin fusion polypeptide. Thevariants of the nucleic acids encoding binding domain-immunoglobulinfusion may be naturally occurring allelic variants of the nucleic acidsor non-naturally occurring variants. As is known in the art, an allelicvariant is an alternate form of a nucleic acid sequence which may haveat least one of a substitution, a deletion or an addition of one or morenucleotides, any of which does not substantially alter the function ofthe encoded binding domain-immunoglobulin fusion polypeptide.

Variants and derivatives of binding domain-immunoglobulin fusion may beobtained by mutations of nucleotide sequences encoding bindingdomain-immunoglobulin fusion polypeptides or any portion thereof.Alterations of the native amino acid sequence may be accomplished by anyof a number of conventional methods. Mutations can be introduced atparticular loci by synthesizing oligonucleotides containing a mutantsequence, flanked by restriction sites enabling ligation to fragments ofthe native sequence. Following ligation, the resulting reconstructedsequence encodes an analog having the desired amino acid insertion,substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene whereinpredetermined codons can be altered by substitution, deletion orinsertion. Exemplary methods of making such alterations are disclosed byWalder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985);Craik (BioTechniques, January 1985, 12-19); Smith et al. (GeneticEngineering: Principles and Methods BioTechniques, January 1985, 12-19);Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press,1981); Kunkel (Proc. Natl. Acad. Sci. USA 82:488, 1985); Kunkel et al.(Methods in Enzymol. 154:367, 1987); and U.S. Pat. Nos. 4,518,584 and4,737,462.

As an example, modification of DNA may be performed by site-directedmutagenesis of DNA encoding the protein combined with the use of DNAamplification methods using primers to introduce and amplify alterationsin the DNA template, such as PCR splicing by overlap extension (SOE).Site-directed mutagenesis is typically effected using a phage vectorthat has single- and double-stranded forms, such as M13 phage vectors,which are well-known and commercially available. Other suitable vectorsthat contain a single-stranded phage origin of replication may be used(see, e.g., Veira et al., Meth. Enzymol. 15:3, 1987). In general,site-directed mutagenesis is performed by preparing a single-strandedvector that encodes the protein of interest (e.g., all or a componentportion of a given binding domain-immunoglobulin fusion protein). Anoligonucleotide primer that contains the desired mutation within aregion of homology to the DNA in the single-stranded vector is annealedto the vector followed by addition of a DNA polymerase, such as E. coliDNA polymerase I (Klenow fragment), which uses the double strandedregion as a primer to produce a heteroduplex in which one strand encodesthe altered sequence and the other the original sequence. Theheteroduplex is introduced into appropriate bacterial cells and clonesthat include the desired mutation are selected. The resulting alteredDNA molecules may be expressed recombinantly in appropriate host cellsto produce the modified protein.

Equivalent DNA constructs that encode various additions or substitutionsof amino acid residues or sequences, or deletions of terminal orinternal residues or sequences not needed for biological activity arealso encompassed by the invention. For example, and as discussed above,sequences encoding Cys residues that are not desirable or essential forbiological activity can be altered to cause the Cys residues to bedeleted or replaced with other amino acids, preventing formation ofincorrect intramolecular disulfide bridges upon renaturation.

Host organisms include those organisms in which recombinant productionof binding domain-immunoglobulin fusion products encoded by therecombinant constructs of the present invention may occur, such asbacteria (for example, E. coli), yeast (for example, Saccharomycescerevisiae and Pichia pastoris), insect cells and mammals, including invitro and in vivo expression. Host organisms thus may include organismsfor the construction, propagation, expression or other steps in theproduction of the compositions provided herein; hosts also includesubjects in which immune responses take place, as described above.Presently preferred host organisms are E. coli bacterial strains, inbredmurine strains and murine cell lines, and human cells, subjects and celllines.

The DNA construct encoding the desired binding domain-immunoglobulinfusion is introduced into a plasmid for expression in an appropriatehost. In preferred embodiments, the host is a bacterial host. Thesequence encoding the ligand or nucleic acid binding domain ispreferably codon-optimized for expression in the particular host. Thus,for example, if a human binding domain-immunoglobulin fusion isexpressed in bacteria, the codons would be optimized for bacterialusage. For small coding regions, the gene can be synthesized as a singleoligonucleotide. For larger proteins, splicing of multipleoligonucleotides, mutagenesis, or other techniques known to those in theart may be used. The sequences of nucleotides in the plasmids that areregulatory regions, such as promoters and operators, are operationallyassociated with one another for transcription. The sequence ofnucleotides encoding a binding domain-immunoglobulin fusion protein mayalso include DNA encoding a secretion signal, whereby the resultingpeptide is a precursor protein. The resulting processed protein may berecovered from the periplasmic space or the fermentation medium.

In preferred embodiments, the DNA plasmids also include a transcriptionterminator sequence. As used herein, a “transcription terminator region”is a sequence that signals transcription termination. The entiretranscription terminator may be obtained from a protein-encoding gene,which may be the same or different from the inserted bindingdomain-immunoglobulin fusion encoding gene or the source of thepromoter. Transcription terminators are optional components of theexpression systems herein, but are employed in preferred embodiments.

The plasmids used herein include a promoter in operative associationwith the DNA encoding the protein or polypeptide of interest and aredesigned for expression of proteins in a suitable host as describedabove (e.g., bacterial, murine or human) depending upon the desired useof the plasmid (e.g., administration of a vaccine containing bindingdomain-immunoglobulin fusion encoding sequences). Suitable promoters forexpression of proteins and polypeptides herein are widely available andare well known in the art. Inducible promoters or constitutive promotersthat are linked to regulatory regions are preferred. Such promotersinclude, but are not limited to, the T7 phage promoter and other T7-likephage promoters, such as the T3, T5 and SP6 promoters, the trp, lpp, andlac promoters, such as the lacUV5, from E. coli; the P10 or polyhedringene promoter of baculovirus/insect cell expression systems (see, e.g.,U.S. Pat. Nos. 5,243,041, 5,242,687, 5,266,317, 4,745,051, and5,169,784) and inducible promoters from other eukaryotic expressionsystems. For expression of the proteins such promoters are inserted in aplasmid in operative linkage with a control region such as the lacoperon.

Preferred promoter regions are those that are inducible and functionalin E. coli. Examples of suitable inducible promoters and promoterregions include, but are not limited to: the E. coli lac operatorresponsive to isopropyl β-D-thiogalactopyranoside (IPTG; see Nakamura etal., Cell 18:1109-1117, 1979); the metallothionein promotermetal-regulatory-elements responsive to heavy-metal (e.g., zinc)induction (see, e.g., U.S. Pat. No. 4,870,009 to Evans et al.); thephage T7lac promoter responsive to IPTG (see, e.g., U.S. Pat. No.4,952,496; and Studier et al., Meth. Enzymol. 185:60-89, 1990) and theTAC promoter.

The plasmids may optionally include a selectable marker gene or genesthat are functional in the host. A selectable marker gene includes anygene that confers a phenotype on bacteria that allows transformedbacterial cells to be identified and selectively grown from among a vastmajority of untransformed cells. Suitable selectable marker genes forbacterial hosts, for example, include the ampicillin resistance gene(Amp^(r)), tetracycline resistance gene (Tc^(r)) and the kanamycinresistance gene (Kan^(r)). The kanamycin resistance gene is presentlypreferred.

The plasmids may also include DNA encoding a signal for secretion of theoperably linked protein. Secretion signals suitable for use are widelyavailable and are well known in the art. Prokaryotic and eukaryoticsecretion signals functional in E. coli may be employed. The presentlypreferred secretion signals include, but are not limited to, thoseencoded by the following E. coli genes: ompA, ompT, ompF, ompC,beta-lactamase, and alkaline phosphatase, and the like (von Heijne, J.Mol. Biol. 184:99-105, 1985). In addition, the bacterial pelB genesecretion signal (Lei et al., J. Bacteriol. 169:4379, 1987), the phoAsecretion signal, and the cek2 functional in insect cell may beemployed. The most preferred secretion signal is the E. coli ompAsecretion signal. Other prokaryotic and eukaryotic secretion signalsknown to those of skill in the art may also be employed (see, e.g., vonHeijne, J. Mol. Biol. 184:99-105, 1985). Using the methods describedherein, one of skill in the art can substitute secretion signals thatare functional in either yeast, insect or mammalian cells to secreteproteins from those cells.

Preferred plasmids for transformation of E. coli cells include the pETexpression vectors (e.g., pET-11a, pET-12a-c, pET-15b; see U.S. Pat. No.4,952,496; available from Novagen, Madison, Wis.). Other preferredplasmids include the pKK plasmids, particularly pKK 223-3, whichcontains the tac promoter (Brosius et al., Proc. Natl. Acad. Sci.81:6929, 1984; Ausubel et al., Current Protocols in Molecular Biology;U.S. Pat. Nos. 5,122,463, 5,173,403, 5,187,153, 5,204,254, 5,212,058,5,212,286, 5,215,907, 5,220,013, 5,223,483, and 5,229,279). Plasmid pKKhas been modified by replacement of the ampicillin resistance gene witha kanamycin resistance gene. (Available from Pharmacia; obtained frompUC4K, see, e.g., Vieira et al. (Gene 19:259-268, 1982; and U.S. Pat.No. 4,719,179.) Baculovirus vectors, such as pBlueBac (also calledpJVETL and derivatives thereof), particularly pBlueBac III (see, e.g.,U.S. Pat. Nos. 5,278,050, 5,244,805, 5,243,041, 5,242,687, 5,266,317,4,745,051, and 5,169,784; available from Invitrogen, San Diego) may alsobe used for expression of the polypeptides in insect cells. Otherplasmids include the pIN-IIIompA plasmids (see U.S. Pat. No. 4,575,013;see also Duffaud et al., Meth. Enz. 153:492-507, 1987), such aspIN-IIIompA2.

Preferably, the DNA molecule is replicated in bacterial cells,preferably in E. coli. The preferred DNA molecule also includes abacterial origin of replication, to ensure the maintenance of the DNAmolecule from generation to generation of the bacteria. In this way,large quantities of the DNA molecule can be produced by replication inbacteria. Preferred bacterial origins of replication include, but arenot limited to, the fl-ori and col E1 origins of replication. Preferredhosts contain chromosomal copies of DNA encoding T7 RNA polymeraseoperably linked to an inducible promoter, such as the lacUV promoter(see U.S. Pat. No. 4,952,496). Such hosts include, but are not limitedto, lysogens E. coli strains HMS174(DE3)pLysS, BL21(DE3)pLysS,HMS174(DE3) and BL21(DE3). Strain BL21(DE3) is preferred. The pLysstrains provide low levels of T7 lysozyme, a natural inhibitor of T7 RNApolymerase.

The DNA molecules provided may also contain a gene coding for arepressor protein. The repressor protein is capable of repressing thetranscription of a promoter that contains sequences of nucleotides towhich the repressor protein binds. The promoter can be derepressed byaltering the physiological conditions of the cell. For example, thealteration can be accomplished by adding to the growth medium a moleculethat inhibits the ability to interact with the operator or withregulatory proteins or other regions of the DNA or by altering thetemperature of the growth media. Preferred repressor proteins include,but are not limited to the E. coli lad repressor responsive to IPTGinduction, the temperature sensitive λ cI857 repressor, and the like.The E. coli lacI repressor is preferred.

In general, recombinant constructs of the subject invention will alsocontain elements necessary for transcription and translation. Inparticular, such elements are preferred where the recombinant expressionconstruct containing nucleic acid sequences encoding bindingdomain-immunoglobulin fusion proteins is intended for expression in ahost cell or organism. In certain embodiments of the present invention,cell type preferred or cell type specific expression of a cell bindingdomain-immunoglobulin fusion encoding gene may be achieved by placingthe gene under regulation of a promoter. The choice of the promoter willdepend upon the cell type to be transformed and the degree or type ofcontrol desired. Promoters can be constitutive or active and may furtherbe cell type specific, tissue specific, individual cell specific, eventspecific, temporally specific or inducible. Cell-type specific promotersand event type specific promoters are preferred. Examples ofconstitutive or nonspecific promoters include the SV40 early promoter(U.S. Pat. No. 5,118,627), the SV40 late promoter (U.S. Pat. No.5,118,627), CMV early gene promoter (U.S. Pat. No. 5,168,062), andadenovirus promoter. In addition to viral promoters, cellular promotersare also amenable within the context of this invention. In particular,cellular promoters for the so-called housekeeping genes are useful.Viral promoters are preferred, because generally they are strongerpromoters than cellular promoters. Promoter regions have been identifiedin the genes of many eukaryotes including higher eukaryotes, such thatsuitable promoters for use in a particular host can be readily selectedby those skilled in the art.

Inducible promoters may also be used. These promoters include MMTV LTR(PCT WO 91/13160), inducible by dexamethasone; metallothionein promoter,inducible by heavy metals; and promoters with cAMP response elements,inducible by cAMP. By using an inducible promoter, the nucleic acidsequence encoding a binding domain-immunoglobulin fusion protein may bedelivered to a cell by the subject invention expression construct andwill remain quiescent until the addition of the inducer. This allowsfurther control on the timing of production of the gene product.

Event-type specific promoters are active or up-regulated only upon theoccurrence of an event, such as tumorigenicity or viral infection. TheHIV LTR is a well known example of an event-specific promoter. Thepromoter is inactive unless the tat gene product is present, whichoccurs upon viral infection. Some event-type promoters are alsotissue-specific.

Additionally, promoters that are coordinately regulated with aparticular cellular gene may be used. For example, promoters of genesthat are coordinately expressed may be used when expression of aparticular binding domain-immunoglobulin fusion protein-encoding gene isdesired in concert with expression of one or more additional endogenousor exogenously introduced genes. This type of promoter is especiallyuseful when one knows the pattern of gene expression relevant toinduction of an immune response in a particular tissue of the immunesystem, so that specific immunocompetent cells within that tissue may beactivated or otherwise recruited to participate in the immune response.

In addition to the promoter, repressor sequences, negative regulators,or tissue-specific silencers may be inserted to reduce non-specificexpression of binding domain-immunoglobulin fusion protein encodinggenes in certain situations, such as, for example, a host that istransiently immunocompromised as part of a therapeutic strategy.Multiple repressor elements may be inserted in the promoter region.Repression of transcription is independent on the orientation ofrepressor elements or distance from the promoter. One type of repressorsequence is an insulator sequence. Such sequences inhibit transcription(Dunaway et al., Mol Cell Biol 17: 182-9, 1997; Gdula et al., Proc NatlAcad Sci USA 93:9378-83, 1996, Chan et al., J Virol 70: 5312-28, 1996;Scott and Geyer, EMBO J 14:6258-67, 1995; Kalos and Fournier, Mol CellRiot 15:198-207, 1995; Chung et al., Cell 74: 505-14, 1993) and willsilence background transcription.

Repressor elements have also been identified in the promoter regions ofthe genes for type II (cartilage) collagen, choline acetyltransferase,albumin (Hu et al., J. Cell Growth Differ. 3(9):577-588, 1992),phosphoglycerate kinase (PGK-2) (Misuno et al., Gene 119(2):293-297,1992), and in the 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatasegene. (Lemaigre et al., Mol. Cell Biol. 11(2):1099-1106.) Furthermore,the negative regulatory element Tse-1 has been identified in a number ofliver specific genes, and has been shown to block cAMP response element-(CRE) mediated induction of gene activation in hepatocytes. (Boshart etal., Cell 61(5):905-916, 1990.)

In preferred embodiments, elements that increase the expression of thedesired product are incorporated into the construct. Such elementsinclude internal ribosome binding sites (IRES; Wang and Siddiqui, Curr.Top. Microbiol. Immunol 203:99, 1995; Ehrenfeld and Semler, Curr. Top.Microbiol. Immunol. 203:65, 1995; Rees et al., Biotechniques 20:102,1996; Sugimoto et al., Biotechnology 12:694, 1994). IRES increasetranslation efficiency. As well, other sequences may enhance expression.For some genes, sequences especially at the 5′ end inhibit transcriptionand/or translation. These sequences are usually palindromes that canform hairpin structures. Any such sequences in the nucleic acid to bedelivered are generally deleted. Expression levels of the transcript ortranslated product are assayed to confirm or ascertain which sequencesaffect expression. Transcript levels may be assayed by any known method,including Northern blot hybridization, RNase probe protection and thelike. Protein levels may be assayed by any known method, includingELISA, western blot, immunocytochemistry or other well known techniques.

Other elements may be incorporated into the bindingdomain-immunoglobulin fusion protein encoding constructs of the presentinvention. In preferred embodiments, the construct includes atranscription terminator sequence, including a polyadenylation sequence,splice donor and acceptor sites, and an enhancer. Other elements usefulfor expression and maintenance of the construct in mammalian cells orother eukaryotic cells may also be incorporated (e.g., origin ofreplication). Because the constructs are conveniently produced inbacterial cells, elements that are necessary for, or that enhance,propagation in bacteria are incorporated. Such elements include anorigin of replication, a selectable marker and the like.

As provided herein, an additional level of controlling the expression ofnucleic acids encoding binding domain-immunoglobulin fusion proteinsdelivered to cells using the constructs of the invention may be providedby simultaneously delivering two or more differentially regulatednucleic acid constructs. The use of such a multiple nucleic acidconstruct approach may permit coordinated regulation of an immuneresponse such as, for example, spatiotemporal coordination that dependson the cell type and/or presence of another expressed encoded component.Those familiar with the art will appreciate that multiple levels ofregulated gene expression may be achieved in a similar manner byselection of suitable regulatory sequences, including but not limited topromoters, enhancers and other well known gene regulatory elements.

The present invention also relates to vectors, and to constructsprepared from known vectors that include nucleic acids of the presentinvention, and in particular to “recombinant expression constructs” thatinclude any nucleic acids encoding binding domain-immunoglobulin fusionproteins and polypeptides according to the invention as provided above;to host cells which are genetically engineered with vectors and/orconstructs of the invention and to methods of administering expressionconstructs comprising nucleic acid sequences encoding such bindingdomain-immunoglobulin fusion polypeptides and fusion proteins of theinvention, or fragments or variants thereof, by recombinant techniques.Binding domain-immunoglobulin fusion proteins can be expressed invirtually any host cell under the control of appropriate promoters,depending on the nature of the construct (e.g., type of promoter, asdescribed above), and on the nature of the desired host cell (e.g.,whether postmitotic terminally differentiated or actively dividing;e.g., whether the expression construct occurs in host cell as an episomeor is integrated into host cell genome). Appropriate cloning andexpression vectors for use with prokaryotic and eukaryotic hosts aredescribed by Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., (1989); as noted above, inparticularly preferred embodiments of the invention, recombinantexpression is conducted in mammalian cells that have been transfected ortransformed with the subject invention recombinant expression construct.

Typically, the constructs are derived from plasmid vectors. A preferredconstruct is a modified pNASS vector (Clontech, Palo Alto, Calif.),which has nucleic acid sequences encoding an ampicillin resistance gene,a polyadenylation signal and a T7 promoter site. Other suitablemammalian expression vectors are well known (see, e.g., Ausubel et al.,1995; Sambrook et al., supra; see also, e.g., catalogues fromInvitrogen, San Diego, Calif.; Novagen, Madison, Wis.; Pharmacia,Piscataway, N.J.; and others). Presently preferred constructs may beprepared that include a dihydrofolate reductase (DHFR) encoding sequenceunder suitable regulatory control, for promoting enhanced productionlevels of the binding domain-immunoglobulin fusion protei, which levelsresult from gene amplification following application of an appropriateselection agent (e.g., methetrexate).

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence, as described above.The heterologous structural sequence is assembled in appropriate phasewith translation initiation and termination sequences. Thus, forexample, the binding domain-immunoglobulin fusion protein encodingnucleic acids as provided herein may be included in any one of a varietyof expression vector constructs as a recombinant expression constructfor expressing a binding domain-immunoglobulin fusion polypeptide in ahost cell. In certain preferred embodiments the constructs are includedin formulations that are administered in vivo. Such vectors andconstructs include chromosomal, nonchromosomal and synthetic DNAsequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA;yeast plasmids; vectors derived from combinations of plasmids and phageDNA, viral DNA, such as vaccinia, adenovirus, fowl pox virus, andpseudorabies, or replication deficient retroviruses as described below.However, any other vector may be used for preparation of a recombinantexpression construct, and in preferred embodiments such a vector will bereplicable and viable in the host.

The appropriate DNA sequence(s) may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Standard techniques for cloning, DNA isolation, amplification andpurification, for enzymatic reactions involving DNA ligase, DNApolymerase, restriction endonucleases and the like, and variousseparation techniques are those known and commonly employed by thoseskilled in the art. A number of standard techniques are described, forexample, in Ausubel et al. (1993 Current Protocols in Molecular Biology,Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., Boston, Mass.);Sambrook et al. (1989 Molecular Cloning, Second Ed., Cold Spring HarborLaboratory, Plainview, N.Y.); Maniatis et al. (1982 Molecular Cloning,Cold Spring Harbor Laboratory, Plainview, N.Y.); Glover (Ed.) (1985 DNACloning Vol. I and II, IRL Press, Oxford, UK); Hames and Higgins (Eds.),(1985 Nucleic Acid Hybridization, IRL Press, Oxford, UK); and elsewhere.

The DNA sequence in the expression vector is operatively linked to atleast one appropriate expression control sequences (e.g., a constitutivepromoter or a regulated promoter) to direct mRNA synthesis.Representative examples of such expression control sequences includepromoters of eukaryotic cells or their viruses, as described above.Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art, and preparation ofcertain particularly preferred recombinant expression constructscomprising at least one promoter or regulated promoter operably linkedto a nucleic acid encoding an binding domain-immunoglobulin fusionpolypeptide is described herein.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 by that act on a promoter to increase itstranscription. Examples include the SV40 enhancer on the late side ofthe replication origin by 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

As provided herein, in certain embodiments the vector may be a viralvector such as a retroviral vector. (Miller et al., 1989 BioTechniques7:980; Coffin and Varmus, 1996 Retroviruses, Cold Spring HarborLaboratory Press, NY.) For example, retroviruses from which theretroviral plasmid vectors may be derived include, but are not limitedto, Moloney Murine Leukemia Virus, spleen necrosis virus, retrovirusessuch as Rous Sarcoma Virus, Harvey Sarcoma virus, avian leukosis virus,gibbon ape leukemia virus, human immunodeficiency virus, adenovirus,Myeloproliferative Sarcoma Virus, and mammary tumor virus.

Retroviruses are RNA viruses which can replicate and integrate into thegenome of a host cell via a DNA intermediate. This DNA intermediate, orprovirus, may be stably integrated into the host cell DNA. According tocertain embodiments of the present invention, an expression constructmay comprise a retrovirus into which a foreign gene that encodes aforeign protein is incorporated in place of normal retroviral RNA. Whenretroviral RNA enters a host cell coincident with infection, the foreigngene is also introduced into the cell, and may then be integrated intohost cell DNA as if it were part of the retroviral genome. Expression ofthis foreign gene within the host results in expression of the foreignprotein.

Most retroviral vector systems which have been developed for genetherapy are based on murine retroviruses. Such retroviruses exist in twoforms, as free viral particles referred to as virions, or as provirusesintegrated into host cell DNA. The virion form of the virus contains thestructural and enzymatic proteins of the retrovirus (including theenzyme reverse transcriptase), two RNA copies of the viral genome, andportions of the source cell plasma membrane containing viral envelopeglycoprotein. The retroviral genome is organized into four main regions:the Long Terminal Repeat (LTR), which contains cis-acting elementsnecessary for the initiation and termination of transcription and issituated both 5′ and 3′ of the coding genes, and the three coding genesgag, pol, and env. These three genes gag, pol, and env encode,respectively, internal viral structures, enzymatic proteins (such asintegrase), and the envelope glycoprotein (designated gp70 and p15e)which confers infectivity and host range specificity of the virus, aswell as the “R” peptide of undetermined function.

Separate packaging cell lines and vector producing cell lines have beendeveloped because of safety concerns regarding the uses of retroviruses,including their use in expression constructs as provided by the presentinvention. Briefly, this methodology employs the use of two components,a retroviral vector and a packaging cell line (PCL). The retroviralvector contains long terminal repeats (LTRs), the foreign DNA to betransferred and a packaging sequence (y). This retroviral vector willnot reproduce by itself because the genes which encode structural andenvelope proteins are not included within the vector genome. The PCLcontains genes encoding the gag, pol, and env proteins, but does notcontain the packaging signal “y”. Thus, a PCL can only form empty virionparticles by itself. Within this general method, the retroviral vectoris introduced into the PCL, thereby creating a vector-producing cellline (VCL). This VCL manufactures virion particles containing only theretroviral vector's (foreign) genome, and therefore has previously beenconsidered to be a safe retrovirus vector for therapeutic use.

“Retroviral vector construct” refers to an assembly which is, withinpreferred embodiments of the invention, capable of directing theexpression of a sequence(s) or gene(s) of interest, such as bindingdomain-immunoglobulin fusion encoding nucleic acid sequences. Briefly,the retroviral vector construct must include a 5′ LTR, a tRNA bindingsite, a packaging signal, an origin of second strand DNA synthesis and a3′ LTR. A wide variety of heterologous sequences may be included withinthe vector construct, including for example, sequences which encode aprotein (e.g., cytotoxic protein, disease-associated antigen, immuneaccessory molecule, or replacement gene), or which are useful as amolecule itself (e.g., as a ribozyme or antisense sequence).

Retroviral vector constructs of the present invention may be readilyconstructed from a wide variety of retroviruses, including for example,B, C, and D type retroviruses as well as spumaviruses and lentiviruses(see, e.g., RNA Tumor Viruses, Second Edition, Cold Spring HarborLaboratory, 1985). Such retroviruses may be readily obtained fromdepositories or collections such as the American Type Culture Collection(“ATCC”; Rockville, Maryland), or isolated from known sources usingcommonly available techniques. Any of the above retroviruses may bereadily utilized in order to assemble or construct retroviral vectorconstructs, packaging cells, or producer cells of the present inventiongiven the disclosure provided herein, and standard recombinanttechniques (e.g., Sambrook et al, Molecular Cloning: A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Kunkle, PNAS82:488, 1985).

Suitable promoters for use in viral vectors generally may include, butare not limited to, the retroviral LTR; the SV40 promoter; and the humancytomegalovirus (CMV) promoter described in Miller, et al.,Biotechniques 7:980-990 (1989), or any other promoter (e.g., cellularpromoters such as eukaryotic cellular promoters including, but notlimited to, the histone, pol III, and β-actin promoters). Other viralpromoters which may be employed include, but are not limited to,adenovirus promoters, thymidine kinase (TK) promoters, and B19parvovirus promoters. The selection of a suitable promoter will beapparent to those skilled in the art from the teachings containedherein, and may be from among either regulated promoters or promoters asdescribed above.

As described above, the retroviral plasmid vector is employed totransduce packaging cell lines to form producer cell lines. Examples ofpackaging cells which may be transfected include, but are not limitedto, the PE501, PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, CRE, ψCRIP,GP+E-86, GP+envAm12, and DAN cell lines as described in Miller, HumanGene Therapy, 1:5-14 (1990). The vector may transduce the packagingcells through any means known in the art. Such means include, but arenot limited to, electroporation, the use of liposomes, and CaPO₄precipitation. In one alternative, the retroviral plasmid vector may beencapsulated into a liposome, or coupled to a lipid, and thenadministered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence(s) encoding the bindingdomain-immunoglobulin fusion polypeptides or fusion proteins. Suchretroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding the bindingdomain-immunoglobulin fusion polypeptide or fusion protein. Eukaryoticcells which may be transduced include, but are not limited to, embryonicstem cells, as well as hematopoietic stem cells, hepatocytes,fibroblasts, circulating peripheral blood mononuclear andpolymorphonuclear cells including myelomonocytic cells, lymphocytes,myoblasts, tissue macrophages, dendritic cells, Kupffer cells, lymphoidand reticuloendothelia cells of the lymph nodes and spleen,keratinocytes, endothelial cells, and bronchial epithelial cells.

As another example of an embodiment of the invention in which a viralvector is used to prepare the recombinant binding domain-immunoglobulinfusion expression construct, in one preferred embodiment, host cellstransduced by a recombinant viral construct directing the expression ofbinding domain-immunoglobulin fusion polypeptides or fusion proteins mayproduce viral particles containing expressed bindingdomain-immunoglobulin fusion polypeptides or fusion proteins that arederived from portions of a host cell membrane incorporated by the viralparticles during viral budding.

In another aspect, the present invention relates to host cellscontaining the above described recombinant binding domain-immunoglobulinfusion expression constructs. Host cells are genetically engineered(transduced, transformed or transfected) with the vectors and/orexpression constructs of this invention which may be, for example, acloning vector, a shuttle vector or an expression construct. The vectoror construct may be, for example, in the form of a plasmid, a viralparticle, a phage, etc. The engineered host cells can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying particular genes suchas genes encoding binding domain-immunoglobulin fusion polypeptides orbinding domain-immunoglobulin fusion proteins. The culture conditionsfor particular host cells selected for expression, such as temperature,pH and the like, will be readily apparent to the ordinarily skilledartisan.

The host cell can be a higher eukaryotic cell, such as a mammalian cell,or a lower eukaryotic cell, such as a yeast cell, or the host cell canbe a prokaryotic cell, such as a bacterial cell. Representative examplesof appropriate host cells according to the present invention include,but need not be limited to, bacterial cells, such as E. coli,Streptomyces, Salmonella tvphimurium; fungal cells, such as yeast;insect cells, such as Drosophila S2 and Spodoptera Sf9; animal cells,such as CHO, COS or 293 cells; adenoviruses; plant cells, or anysuitable cell already adapted to in vitro propagation or so establishedde novo. The selection of an appropriate host is deemed to be within thescope of those skilled in the art from the teachings herein.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell23:175 (1981), and other cell lines capable of expressing a compatiblevector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.Mammalian expression vectors will comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation site, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking nontranscribedsequences, for example as described herein regarding the preparation ofbinding domain-immunoglobulin fusion expression constructs. DNAsequences derived from the SV40 splice, and polyadenylation sites may beused to provide the required nontranscribed genetic elements.Introduction of the construct into the host cell can be effected by avariety of methods with which those skilled in the art will be familiar,including but not limited to, for example, calcium phosphatetransfection, DEAE-Dextran mediated transfection, or electroporation(Davis et al., 1986 Basic Methods in Molecular Biology).

The present invention binding domain-immunoglobulin fusion proteins, orcompositions comprising one or more polynucleotides encoding same asdescribed herein, (for example, to be administered under conditions andfor a time sufficient to permit expression of a bindingdomain-immunoglobulin fusion protein in a host cell in vivo or invitro), may be formulated into pharmaceutical compositions foradministration according to well known methodologies. Pharmaceuticalcompositions generally comprise one or more recombinant expressionconstructs, and/or expression products of such constructs, incombination with a pharmaceutically acceptable carrier, excipient ordiluent. Such carriers will be nontoxic to recipients at the dosages andconcentrations employed. For nucleic acid-based formulations, or forformulations comprising expression products of the subject inventionrecombinant constructs, about 0.01 μg/kg to about 100 mg/kg body weightwill be administered, typically by the intradermal, subcutaneous,intramuscular or intravenous route, or by other routes. A preferreddosage is about 1 μg/kg to about 1 mg/kg, with about 5 μg/kg to about200 μg/kg particularly preferred. It will be evident to those skilled inthe art that the number and frequency of administration will bedependent upon the response of the host. “Pharmaceutically acceptablecarriers” for therapeutic use are well known in the pharmaceutical art,and are described, for example, in Remingtons Pharmaceutical Sciences,Mack Publishing Co. (A. R. Gennaro edit. 1985). For example, sterilesaline and phosphate-buffered saline at physiological pH may be used.Preservatives, stabilizers, dyes and even flavoring agents may beprovided in the pharmaceutical composition. For example, sodiumbenzoate, sorbic acid and esters of p-hydroxybenzoic acid may be addedas preservatives. Id. at 1449. In addition, antioxidants and suspendingagents may be used. Id.

“Pharmaceutically acceptable salt” refers to salts of the compounds ofthe present invention derived from the combination of such compounds andan organic or inorganic acid (acid addition salts) or an organic orinorganic base (base addition salts). The compounds of the presentinvention may be used in either the free base or salt forms, with bothforms being considered as being within the scope of the presentinvention.

The pharmaceutical compositions that contain one or more bindingdomain-immunoglobulin fusion protein encoding constructs (or theirexpressed products) may be in any form which allows for the compositionto be administered to a patient. For example, the composition may be inthe form of a solid, liquid or gas (aerosol). Typical routes ofadministration include, without limitation, oral, topical, parenteral(e.g., sublingually or buccally), sublingual, rectal, vaginal, andintranasal. The term parenteral as used herein includes subcutaneousinjections, intravenous, intramuscular, intrasternal, intracavernous,intrathecal, intrameatal, intraurethral injection or infusiontechniques. The pharmaceutical composition is formulated so as to allowthe active ingredients contained therein to be bioavailable uponadministration of the composition to a patient. Compositions that willbe administered to a patient take the form of one or more dosage units,where for example, a tablet may be a single dosage unit, and a containerof one or more compounds of the invention in aerosol form may hold aplurality of dosage units.

For oral administration, an excipient and/or binder may be present.Examples are sucrose, kaolin, glycerin, starch dextrins, sodiumalginate, carboxymethylcellulose and ethyl cellulose. Coloring and/orflavoring agents may be present. A coating shell may be employed.

The composition may be in the form of a liquid, e.g., an elixir, syrup,solution, emulsion or suspension. The liquid may be for oraladministration or for delivery by injection, as two examples. Whenintended for oral administration, preferred compositions contain, inaddition to one or more binding domain-immunoglobulin fusion constructor expressed product, one or more of a sweetening agent, preservatives,dye/colorant and flavor enhancer. In a composition intended to beadministered by injection, one or more of a surfactant, preservative,wetting agent, dispersing agent, suspending agent, buffer, stabilizerand isotonic agent may be included.

A liquid pharmaceutical composition as used herein, whether in the formof a solution, suspension or other like form, may include one or more ofthe following adjuvants: sterile diluents such as water for injection,saline solution, preferably physiological saline, Ringer's solution,isotonic sodium chloride, fixed oils such as synthetic mono ordigylcerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The parenteral preparation can be enclosedin ampoules, disposable syringes or multiple dose vials made of glass orplastic. Physiological saline is a preferred adjuvant. An injectablepharmaceutical composition is preferably sterile.

It may also be desirable to include other components in the preparation,such as delivery vehicles including but not limited to aluminum salts,water-in-oil emulsions, biodegradable oil vehicles, oil-in-wateremulsions, biodegradable microcapsules, and liposomes. Examples ofimmunostimulatory substances (adjuvants) for use in such vehiclesinclude N-acetylmuramyl-L-alanine-D-isoglutamine (MDP),lipopoly-saccharides (LPS), glucan, IL-12, GM-CSF, gamma interferon andIL-15.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will vary depending on the mode of administrationand whether a sustained release is desired. For parenteraladministration, such as subcutaneous injection, the carrier preferablycomprises water, saline, alcohol, a fat, a wax or a buffer. For oraladministration, any of the above carriers or a solid carrier, such asmannitol, lactose, starch, magnesium stearate, sodium saccharine,talcum, cellulose, glucose, sucrose, and magnesium carbonate, may beemployed. Biodegradable microspheres (e.g., polylactic galactide) mayalso be employed as carriers for the pharmaceutical compositions of thisinvention. Suitable biodegradable microspheres are disclosed, forexample, in U.S. Pat. Nos. 4,897,268 and 5,075,109. In this regard, itis preferable that the microsphere be larger than approximately 25microns.

Pharmaceutical compositions may also contain diluents such as buffers,antioxidants such as ascorbic acid, low molecular weight (less thanabout 10 residues) polypeptides, proteins, amino acids, carbohydratesincluding glucose, sucrose or dextrins, chelating agents such as EDTA,glutathione and other stabilizers and excipients. Neutral bufferedsaline or saline mixed with nonspecific serum albumin are exemplaryappropriate diluents. Preferably, product is formulated as alyophilizate using appropriate excipient solutions (e.g., sucrose) asdiluents.

As described above, the subject invention includes compositions capableof delivering nucleic acid molecules encoding bindingdomain-immunoglobulin fusion proteins. Such compositions includerecombinant viral vectors (e.g., retroviruses (see WO 90/07936, WO91/02805, WO 93/25234, WO 93/25698, and WO 94/03622), adenovirus (seeBerkner, Biotechniques 6:616-627, 1988; Li et al., Hum. Gene Ther.4:403-409, 1993; Vincent et al., Nat. Genet. 5:130-134, 1993; and Kollset al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994), pox virus (seeU.S. Pat. No. 4,769,330; U.S. Pat. No. 5,017,487; and WO 89/01973)),recombinant expression construct nucleic acid molecules complexed to apolycationic molecule (see WO 93/03709), and nucleic acids associatedwith liposomes (see Wang et al., Proc. Natl. Acad. Sci. USA 84:7851,1987). In certain embodiments, the DNA may be linked to killed orinactivated adenovirus (see Curiel et al., Hum. Gene Ther. 3:147-154,1992; Cotton et al., Proc. Natl. Acad. Sci. USA 89:6094, 1992). Othersuitable compositions include DNA-ligand (see Wu et al., J. Biol. Chem.264:16985-16987, 1989) and lipid-DNA combinations (see Felgner et al.,Proc. Natl. Acad. Sci. USA 84:7413-7417, 1989).

In addition to direct in vivo procedures, ex vivo procedures may be usedin which cells are removed from a host, modified, and placed into thesame or another host animal. It will be evident that one can utilize anyof the compositions noted above for introduction of bindingdomain-immunoglobulin fusion proteins or of bindingdomain-immunoglobulin fusion protein encoding nucleic acid moleculesinto tissue cells in an ex vivo context. Protocols for viral, physicaland chemical methods of uptake are well known in the art.

Accordingly, the present invention is useful for treating a patienthaving a B-cell disorder or a malignant condition, or for treating acell culture derived from such a patient. As used herein, the term“patient” refers to any warm-blooded animal, preferably a human. Apatient may be afflicted with cancer or a malignant condition, such asB-cell lymphoma, or may be normal (i.e., free of detectable disease andinfection). A “cell culture” includes any preparation amenable to exvivo treatment, for example a preparation containing immunocompetentcells or isolated cells of the immune system (including, but not limitedto, T cells, macrophages, monocytes, B cells and dendritic cells). Suchcells may be isolated by any of a variety of techniques well known tothose of ordinary skill in the art (e.g., Ficoll-hypaque densitycentrifugation). The cells may (but need not) have been isolated from apatient afflicted with a B-cell disorder or a malignant condition, andmay be reintroduced into a patient after treatment.

A liquid composition intended for either parenteral or oraladministration should contain an amount of binding domain-immunoglobulinfusion protein encoding construct or expressed product such that asuitable dosage will be obtained. Typically, this amount is at least0.01 wt % of a binding domain-immunoglobulin fusion construct orexpressed product in the composition. When intended for oraladministration, this amount may be varied to be between 0.1 and about70% of the weight of the composition. Preferred oral compositionscontain between about 4% and about 50% of binding domain-immunoglobulinfusion construct or expressed product(s). Preferred compositions andpreparations are prepared so that a parenteral dosage unit containsbetween 0.01 to 1% by weight of active compound.

The pharmaceutical composition may be intended for topicaladministration, in which case the carrier may suitably comprise asolution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, beeswax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device. Topical formulations may contain aconcentration of the binding domain-immunoglobulin fusion construct orexpressed product of from about 0.1 to about 10% w/v (weight per unitvolume).

The composition may be intended for rectal administration, in the form,e.g., of a suppository which will melt in the rectum and release thedrug. The composition for rectal administration may contain anoleaginous base as a suitable nonirritating excipient. Such basesinclude, without limitation, lanolin, cocoa butter and polyethyleneglycol.

In the methods of the invention, the binding domain-immunoglobulinfusion encoding constructs or expressed product(s) may be administeredthrough use of insert(s), bead(s), timed-release formulation(s),patch(es) or fast-release formulation(s).

The following Examples are offered by way of illustration and not by wayof limitation.

Examples Example 1 Cloning OF THE 2H7 Variable Regions and Constructionand Sequencing of 2H7SCFV-IG

This Example illustrates the cloning of cDNA molecules that encode theheavy chain and light chain variable regions of the monoclonal antibody2H7. This Example also demonstrates the construction, sequencing, andexpression of 2H7scFv-Ig.

Hybridoma cells expressing 2H7 monoclonal antibody that specificallybound to CD20 were provided by Ed Clark at the University of Washington,Seattle, Wash. Prior to harvesting, hybridoma cells were kept in logphase growth for several days in RPMI 1640 media Invitrogen/LifeTechnologies, Gaithersburg, Md.) supplemented with glutamine, pyruvate,DMEM non-essential amino acids, and penicillin-streptomycin. Cells werepelleted by centrifugation from the culture medium, and 2×10⁷ cells wereused to prepare RNA. RNA was isolated from the 2H7-producing hybridomacells using the Pharmingen (San Diego, Calif.) total RNA isolation kit(Catalog #45520K) according to the manufacturer's instructionsaccompanying the kit. One microgram (1 μg) of total RNA was used astemplate to prepare cDNA by reverse transcription. The RNA and 300 ngrandom primers were combined and denatured at 72° C. for 10 minutesprior to addition of enzyme. Superscript II reverse transcriptase (LifeTechnologies) was added to the RNA plus primer mixture in a total volumeof 25 μl in the presence of 5× second strand buffer and 0.1 M DTTprovided with the enzyme. The reverse transcription reaction was allowedto proceed at 42° C. for one hour.

The 2H7 cDNA generated in the randomly primed reverse transcriptasereaction and V region specific primers were used to amplify by PCR thevariable regions for the light and heavy chain of the 2H7 antibody. TheV region specific primers were designed using the published sequence(Genbank accession numbers M17954 for V_(L) and M17953 for V_(H)) as aguide. The two variable chains were designed with compatible endsequences so that an scFv could be assembled by ligation of the two Vregions after amplification and restriction enzyme digestion.

A (gly₄ser)₃ peptide linker to be inserted between the two V regions wasincorporated by adding the extra nucleotides to the antisense primer forthe V_(L) of 2H7. A Sac I restriction site was also introduced at thejunction between the two V regions. The sense primer used to amplify the2H7 V_(L), that included a HindIII restriction site and the light chainleader peptide was 5′-gtc aag ctt gcc gcc atg gat ttt caa gtg cag attttt cag c-3′ (SEQ ID NO: 23). The antisense primer was 5′-gtc gtc gagctc cca cct cct cca gat cca cca ccg ccc gag cca ccg cca cct ttc agc tccagc ttg gtc cc-3″ (SEQ ID NO: 24). The reading frame of the V region isindicated as a bold, underlined codon. The Hind III and Sad sites areindicated by underlined italicized sequences.

The V_(H) domain was amplified without a leader peptide, but included a5′ SacI restriction site for fusion to the V_(L) and a BclI restrictionsite at the 3′ end for fusion to various tails, including the human IgG1Fc domain and the truncated forms of CD40 ligand, CD154. The senseprimer was 5′-gct get gag ctc tca ggc tta tct aca gca agt ctg g-3′ (SEQID NO: 25). The Sad site is indicated in italicized and underlined font,and the reading frame of the codon for the first amino acid of the V_(H)domain is indicated in bold, underlined type. The antisense primer was5′-gtt gtc tga tca gag acg gtg acc gtg gtc cc-3′ (SEQ ID NO: 26). TheBclI site is indicated in italicized, underlined type, and the lastserine of the V_(H) domain sequence is indicated in bold, underlinedtype.

The scFv-Ig was assembled by inserting the 2H7 scFv HindIII-BclIfragment into pUC19 containing the human IgG1 hinge, CH2, and CH3regions, which was digested with restriction enzymes, HindIII and BclI.After ligation, the ligation products were transformed into DH5αbacteria. Positive clones were screened for the properly insertedfragments using the Sad site at the V_(L)-V_(H) junction of 2H7 as adiagnostic site. The 2H7scFv-Ig cDNA was subjected to cycle sequencingon a PE 9700 thermocycler using a 25-cycle program by denaturing at 96°C. for 10 seconds, annealing at 50° C. for 30 seconds, and extending at72° C. for 4 minutes. The sequencing primers were pUC forward andreverse primers and an internal primer that annealed to the CH2 domainhuman in the IgG constant region portion. Sequencing reactions wereperformed using the Big Dye Terminator Ready Sequencing Mix (PE-AppliedBiosystems, Foster City, Calif.) according to the manufacturer'sinstructions. Samples were subsequently purified using Centrisep columns(Catalog #CS-901, Princeton Separations, Adelphia, N.J.), the eluatesdried in a Savant vacuum dryer, denatured in Template SuppressionReagent (PE-ABI), and analyzed on an ABI 310 Genetic Analyzer(PE-Applied Biosystems). The sequence was edited, translated, andanalyzed using Vector Nti version 6.0 (Informax, North Bethesda, Md.).FIG. 1 shows the cDNA and predicted amino acid sequence of the2H7scFv-Ig construct.

Example 2 Expression of 2H7 ScFv-IG in Stable CHO Cell Lines

This Example illustrates expression of 2H7scFv-Ig in a eukaryotic cellline and characterization of the expressed 2H7scFv-Ig by SDS-PAGE and byfunctional assays, including ADCC and complement fixation.

The 2H7scFv-Ig HindIII-XbaI (˜1.6 kb) fragment with correct sequence wasinserted into the mammalian expression vector pD18, and DNA frompositive clones was amplified using QIAGEN plasmid preparation kits(QIAGEN, Valencia, Calif.). The recombinant plasmid DNA (100 μg) wasthen linearized in a nonessential region by digestion with AscI,purified by phenol extraction, and resuspended in tissue culture media,Excell 302 (Catalog #14312-79P, JRH Biosciences, Lenexa, Kans.). Cellsfor transfection, CHO DG44 cells, were kept in logarithmic growth, and10⁷ cells harvested for each transfection reaction. Linearized DNA wasadded to the CHO cells in a total volume of 0.8 ml for electroporation.

Stable production of the 2H7 scFv-Ig fusion protein (SEQ. ID NO:10) wasachieved by electroporation of a selectable, amplifiable plasmid, pD18,containing the 2H7 scFv-Ig cDNA under the control of the CMV promoter,into Chinese Hamster Ovary (CHO) cells (all cell lines from AmericanType Culture Collection, Manassas, Va., unless otherwise noted). The 2H7expression cassette was subcloned downstream of the CMV promoter intothe vector multiple cloning site as a ˜1.6 kb HindIII-XbaI fragment. ThepD18 vector is a modified version of pcDNA3 encoding the DHFR selectablemarker with an attenuated promoter to increase selection pressure forthe plasmid. Plasmid DNA was prepared using Qiagen maxiprep kits, andpurified plasmid was linearized at a unique AscI site prior to phenolextraction and ethanol precipitation. Salmon sperm DNA (Sigma-Aldrich,St. Louis, Mo.) was added as carrier DNA, and 100 μg each of plasmid andcarrier DNA was used to transfect 10⁷ CHO DG44 cells by electroporation.Cells were grown to logarithmic phase in Excell 302 media (JRHBiosciences) containing glutamine (4 mM), pyruvate, recombinant insulin,penicillin-streptomycin, and 2× DMEM nonessential amino acids (all fromLife Technologies, Gaithersburg, Md.), hereafter referred to as “Excell302 complete” media. Media for untransfected cells also contained HT(diluted from a 100× solution of hypoxanthine and thymidine)(Invitrogen/Life Technologies). Media for transfections under selectioncontained varying levels of methotrexate (Sigma-Aldrich) as selectiveagent, ranging from 50 nM to 5 μM. Electroporations were performed at275 volts, 950 μF. Transfected cells were allowed to recover overnightin non-selective media prior to selective plating in 96 well flat bottomplates (Costar) at varying serial dilutions ranging from 125 cells/wellto 2000 cells/well. Culture media for cell cloning was Excell 302complete, containing 100 nM methotrexate. Once clonal outgrowth wassufficient, serial dilutions of culture supernatants from master wellswere screened for binding to CD20-CHO transfected cells. The clones withthe highest production of the fusion protein were expanded into T25 andthen T75 flasks to provide adequate numbers of cells for freezing andfor scaling up production of the 2H7scFvIg. Production levels werefurther increased in cultures from three clones by progressiveamplification in methotrexate containing culture media. At eachsuccessive passage of cells, the Excell 302 complete media contained anincreased concentration of methotrexate, such that only the cells thatamplified the DHFR plasmid could survive.

Supernatants were collected from CHO cells expressing the 2H7scFv-Ig,filtered through 0.2 μm PES express filters (Nalgene, Rochester, N.Y.)and were passed over a Protein A-agarose (IPA 300 crosslinked agarose)column (Repligen, Needham, Mass.). The column was washed with PBS, andthen bound protein was eluted using 0.1 M citrate buffer, pH 3.0.Fractions were collected and eluted protein was neutralized using 1MTris, pH 8.0, prior to dialysis overnight in PBS. Concentration of thepurified 2H7scFv-Ig (SEQ ID NOs: 1 and 11) was determined by absorptionat 280 nm. An extinction coefficient of 1.77 was determined using theprotein analysis tools in the Vector Nti Version 6.0 Software package(Informax, North Bethesda, Md.). This program uses the amino acidcomposition data to calculate extinction coefficients.

Production levels of 2H7scFv-Ig by transfected, stable CHO cells wereanalyzed by flow cytometry. Purified 2H7scFv-Ig to CHO cells was allowedto bind to CHO cells that expressed CD20 (CD20 CHO) and analyzed by flowcytometry using a fluorescein-conjugated anti-human IgG second stepreagent (Catalog Numbers H10101 and H10501, CalTag, Burlingame, Calif.).FIG. 2 (top) shows a standard curve generated by titration of 2H7scFv-Igbinding to CD20 CHO. At each concentration of 2H7scFv-Ig, the meanbrightness of the fluorescein signal in linear units is shown.Supernatants collected from T flasks containing stable CHO cell clonesexpressing 2H7scFv-Ig were then allowed to bind to CD20 CHO and thebinding was analyzed by flow cytometry. The fluorescein signal generatedby 2H7scFv-Ig contained in the supernatants was measured and the2H7scFv-Ig concentration in the supernatants was calculated from thestandard curve (FIG. 2, bottom).

Purified 2H7scFv-Ig (SEQ ID NOs: 1 and 11) was analyzed byelectrophoresis on SDS-Polyacrylamide gels. Samples of 2H7scFv-Ig,purified by independent Protein A Agarose column runs, were boiled inSDS sample buffer without reduction of disulfide bonds and applied toSDS 10% Tris-BIS gels (Catalog #NP0301, Novex, Carlsbad, Calif.). Twentymicrograms of each purified batch was loaded on the gels. The proteinswere visualized after electrophoresis by Coomassie Blue staining (PierceGel Code Blue Stain Reagent, Catalog #24590, Pierce, Rockford, Ill.),and destaining in distilled water. Molecular weight markers wereincluded on the same gel (Kaleidoscope Prestained Standards, Catalog#161-0324, Bio-Rad, Hercules, Calif.). The results are presented in FIG.3. The numbers above the lanes designate independent purificationbatches. The molecular weights in kilodaltons of the size markers areindicated on the left side of the figure. Further experiments withalternative sample preparation conditions indicated that reduction ofdisulfide bonds by boiling the protein in SDS sample buffer containingDTT or 2-mercaptoethanol caused the 2H7scFv-Ig to aggregate.

Any number of other immunological parameters may be monitored usingroutine assays that are well known in the art. These may include, forexample, antibody dependent cell-mediated cytotoxicity (ADCC) assays,secondary in vitro antibody responses, flow immunocytofluorimetricanalysis of various peripheral blood or lymphoid mononuclear cellsubpopulations using well established marker antigen systems,immunohistochemistry or other relevant assays. These and other assaysmay be found, for example, in Rose et al. (Eds.), Manual of ClinicalLaboratory Immunology, 5^(th) Ed., 1997 American Society ofMicrobiology, Washington, D.C.

The ability of 2H7scFv-Ig to kill CD20 positive cells in the presence ofcomplement was tested using B cell lines Ramos and Bjab. Rabbitcomplement (Pel-Freez, Rogers, Ark.) was used in the assay at a finaldilution of 1/10. Purified 2H7scFv-Ig was incubated with B cells andcomplement for 45 minutes at 37° C., followed by counting of live anddead cells by trypan blue exclusion. The results in FIG. 4A show that inthe presence of rabbit complement, 2H7scFv-Ig lysed B cells expressingCD20.

The ability of 2H7scFv-Ig to kill CD20 positive cells in the presence ofperipheral blood mononuclear cells (PBMC) was tested by measuring therelease of ⁵¹Cr from labeled Bjab cells in a 4-hour assay using a 100:1ratio of PBMC to Bjab cells. The results shown in FIG. 4B indicated that2H7scFv-Ig can mediate antibody dependent cellular cytotoxicity (ADCC)because the release of ⁵¹Cr was higher in the presence of both PBMC and2H7scFv-Ig than in the presence of either PBMC or 2H7scFv-Ig alone.

Example 3 Effect of Simultaneous Ligation of CD20 and CD40 on Growth ofNormal B Cells, and on CD95 Expression, and induction of Apoptosis

This example illustrates the effect of cross-linking of CD20 and CD40expressed on the cell surface on cell proliferation.

Dense resting B cells were isolated from human tonsil by a Percoll stepgradient and T cells were removed by E-rosetting. Proliferation ofresting, dense tonsillar B cells was measured by uptake of³[H]-thymidine during the last 12 hours of a 4-day experiment.Proliferation was measured in quadruplicate cultures with means andstandard deviations as shown. Murine anti-human CD20 mAb 1F5 (anti-CD20)was used alone or was cross-linked with anti-murine κ mAb 187.1(anti-CD20XL). CD40 activation was accomplished using soluble humanCD154 fused with murine CD8 (CD154) (Hollenbaugh et al., EMBO J. 11:4212-21 (1992)), and CD40 cross-linking was accomplished usinganti-murine CD8 mAb 53-6 (CD154XL). This procedure allowed simultaneouscross-linking of CD20 and CD40 on the cell surface. The results arepresented in FIG. 5.

The effect of CD20 and CD40 cross-linking on Ramos cells, a B lymphomacell line, was examined. Ramos cells were analyzed for CD95 (Fas)expression and percent apoptosis eighteen hours after treatment (no goatanti-mouse IgG (GAM)) and/or cross-linking (+GAM) using murine mAbs thatspecifically bind CD20 (1F5) and CD40 (G28-5). Control cells weretreated with a non-binding isotype control (64.1) specific for CD3.

Treated Ramos cells were harvested, incubated with FITC-anti-CD95, andanalyzed by flow cytometry to determine the relative expression level ofFas on the cell surface after CD20 or CD40 cross-linking Data is plottedas mean fluorescence of cells after treatment with the stimuli indicated(FIG. 6A).

Treated Ramos cells from the same experiment were harvested and bindingof annexin V was measured to indicate the percentage apoptosis in thetreated cultures. Apoptosis was measured by binding of Annexin V 18hours after cross-linking of CD20 and CD40 using 1F5 and G28-5 followedby cross-linking with GAM. Binding of Annexin V was measured using aFITC-Annexin V kit (Catalog #PN-IM2376, Immunotech, Marseille, France,)Annexin V binding is known to be an early event in progression of cellsinto apoptosis. Apoptosis, or programmed cell death, is a processcharacterized by a cascade of catabolic reactions leading to cell deathby suicide. In the early phase of apoptosis, before cells changemorphology and hydrolyze DNA, the integrity of the cell membrane ismaintained but cells lose the asymmetry of their membrane phospholipids,exposing negatively charged phospholipids, such as phosphatidylserine,at the cell surface. Annexin V, a calcium and phopholipid bindingprotein, binds preferentially and with high affinity tophosphatidylserine. Results demonstrating the effect of cross-linkingboth CD20 and CD40 on expression of the FAS receptor (CD95) arepresented in FIG. 6B. The effect of cross-linking of both CD20 and CD40on Annexin V binding to cells is shown in FIG. 6B.

Example 4 Construction and Characterization of 2H7 SCFv-CD 154 FusionProteins

To construct a molecule capable of binding to both CD20 and CD40, cDNAencoding the 2H7 scFv was fused with cDNA encoding CD154, the CD40ligand. The 2H7 scFv cDNA encoded on the HindIII-BclI fragment wasremoved from the 2H7 scFvIg construct, and inserted into a pD18 vectoralong with a BamHI-XbaI cDNA fragment encoding the extracellular domainof human CD154. The extracellular domain is encoded at the carboxyterminus of CD154, similar to other type II membrane proteins.

The extracellular domain of human CD154 was PCR amplified using cDNAgenerated with random primers and RNA from human T lymphocytes activatedwith PHA (phytohemagglutinin) The primer sets included two different 5′or sense primers that created fusion junctions at two differentpositions within the extracellular domain of CD154. Two different fusionjunctions were designed that resulted in a short or truncated form (formS4) including amino acids 108 (Glu)-261 (Leu)+(Glu), and a long orcomplete form (form L2) including amino acids 48 (Arg) -261 (Leu)+(Glu),of the extracellular domain of CD154, both constructed as BamHI-XbaIfragments. The sense primer which fuses the two different truncatedextracellular domains to the 2H7scFv includes a BamHI site for cloning.The sense primer for the S4 form of the CD154 cDNA is designatedCD154BAM108 and encodes a 34 mer with the following sequence: 5′-gtt gtcgga tcc aga aaa cag ctt tga aat gca a-3′ (SEQ ID NO: 27), while theantisense primer is designated CD154XBA and encodes a 44 mer with thefollowing sequence: 5′-gtt gtt tct aga tta tca ctc gag ttt gag taa gccaaa gga cg-3′ (SEQ ID NO: 28).

The oligonucleotide primers used in amplifying the long form (L2) of theCD154 extracellular domain encoding amino acids 48 (Arg)-261(Leu)+(Glu), were as follows: The sense primer designated CD154 BAM48encoded a 35-mer with the following sequence: 5′-gtt gtc gga tcc aag aaggtt gga caa gat aga ag-3′(SEQ ID NO: 29). The antisense primerdesignated CD154XBA encoded the 44-mer: 5′-gtt gtt tct aga tta tca ctcgag ttt gag taa gcc aaa gga cg-3′ (SEQ ID NO: 28). Other PCR reactionconditions were identical to those used for amplifying the 2H7 scFv (seeExample 1). PCR fragments were purified by PCR quick kits (QIAGEN, SanDiego, Calif.), eluted in 30 μl ddH₂O, and digested with BamHI and XbaI(Roche) restriction endonucleases in a 40 μl reaction volume at 37° C.for 3 hours. Fragments were gel purified, purified using QIAEX kitsaccording to the manufacturer's instructions (QIAGEN), and ligated alongwith the 2H7 HindIII-BclI fragment into the pD18 expression vectordigested with HindIII+XbaI. Ligation reactions were transformed intoDH5-alpha chemically competent bacteria and plated onto LB platescontaining 100 μg/ml ampicillin. Transformants were grown overnight at37° C., and isolated colonies used to inoculate 3 ml liquid cultures inLuria Broth containing 100 μg/ml ampicillin. Clones were screened aftermini-plasmid preparations (QIAGEN) for insertion of both the 2H7 scFvand the CD 154 extracellular domain fragments.

The 2H7scFv-CD154 construct cDNAs were subjected to cycle sequencing ona PE 9700 thermocycler using a 25-cycle program that includeddenaturating at 96° C., 10 seconds, annealing at 50° C. for 5 seconds,and extension at 60° C., for 4 minutes. The sequencing primers used werepD18 forward (SEQ ID NO: 30: 5′-gtctatataagcagagctctggc-3′) and pD18reverse (SEQ ID NO: 31: 5′-cgaggctgatcagcgagctctagca-3′) primers. Inaddition, an internal primer was used that had homology to the humanCD154 sequence (SEQ ID NO: 32: 5′-ccgcaatttgaggattctgatcacc-3′).Sequencing reactions included primers at 3.2 pmol, approximately 200 ngDNA template, and 8 μl sequencing mix. Sequencing reactions wereperformed using the Big Dye Terminator Ready Sequencing Mix (PE-AppliedBiosystems, Foster City, Calif.) according to the manufacturer'sinstructions. Samples were subsequently purified using Centrisep columns(Princeton Separations, Adelphia, N.J.). The eluates were dried in aSavant speed-vacuum dryer, denatured in 20 μl template SuppressionReagent (ABI) at 95° C. for 2 minutes, and analyzed on an ABI 310Genetic Analyzer (PE-Applied Biosystems). The sequence was edited,translated, and analyzed using Vector Nti version 6.0 (Informax, NorthBethesda, Md.). The 2H7scFv-CD 154 L2 cDNA sequence (SEQ ID NOs: 21 and149) and predicted amino acid sequence (SEQ ID NOs: 33 and 150) ispresented in FIG. 7A, and 2H7scFv-CD154 S4 cDNA sequence (SEQ ID NOs: 22and 151) and predicted amino acid sequence (SEQ ID NOs: 34 and 152) ispresented in FIG. 7B.

The binding activity of the 2H7 scFv-CD154 fusion proteins (SEQ ID NOS:33, 150 and 34, 152) to CD20 and CD40 simultaneously was determined byflow cytometry. The assay used CHO cell targets that express CD20. Aftera 45-minute incubation of CD20 CHO cells with supernatants from cellstransfected with the 2H7 scFv-CD154 expression plasmid, the CD20 CHOcells were washed twice and incubated with biotin-conjugated CD40-Igfusion protein in PBS/2% FBS. After 45 min, cells were washed twice andincubated with phycoerythrin (PE)-labeled strepavidin at 1:100 in PBS/2%FBS (Molecular Probes, Eugene OR). After an additional 30 minincubation, cells were washed 2× and were analyzed by flow cytometry.The results show that the 2H7 scFv-CD154 molecule was able to bind toCD20 on the cell surface and to capture biotin-conjugated CD40 fromsolution (FIG. 8).

To determine the effect of the 2H7scFv-CD154 on growth and viability ofB lymphoma and lymphoblastoid cell lines, cells were incubated with2H7scFv-CD154 L2 (SEQ. ID NO: 33, 150) for 12 hours and then examinedfor binding of Annexin V. Binding of Annexin V was measured using aFITC-Annexin V kit (Immunotech, Marseille, France, Catalog #PN-IM2376).B cell lines were incubated in 1 ml cultures with dilutions ofconcentrated, dialyzed supernatants from cells expressing secreted formsof the 2H7scFv-CD154 fusion proteins. The results are presented in FIG.9.

The growth rate of the Ramos B lymphoma cell line in the presence of2H7scFv-CD154 was examined by uptake of ³H-thymidine for the last 6hours of a 24-hour culture. The effect of 2H7scFv-CD154 on cellproliferation is shown in FIG. 10.

Example 5 Construction and Characterization of CytoxB AntibodyDerivatives

CytoxB antibodies were derived from the 2H7 scFv-IgG polypeptide. The2H7 scFv (see Example 1) was linked to the human IgG1 Fc domain via analtered hinge domain (see FIG. 11). Cysteine residues in the hingeregion were substituted with serine residues by site-directedmutagenesis and other methods known in the art. The mutant hinge wasfused either to a wild-type Fc domain to create one construct,designated CytoxB-MHWTG1C, or was fused to a mutated Fc domain(CytoxB-MHMG1C) that had additional mutations introduced into the CH2domain. Amino acid residues in CH2 that are implicated in effectorfunction are illustrated in FIG. 11. Mutations of one or more of theseresidues may reduce FcR binding and mediation of effector functions. Inthis example, the leucine residue 234 known in the art to be importantto Fc receptor binding, was mutated in the 2H7 scFv fusion protein,CytoxB-[MG1H/MG1C]. In another construct, the human IgG1 hinge regionwas substituted with a portion of the human IgA hinge, which was fusedto wild-type human Fc domain (CytoxB-IgAHWTHG1C). (See FIG. 11.) Thismutated hinge region allows expression of a mixture of monomeric anddimeric molecules that retain functional properties of the human IgG1CH2 and CH3 domains. Synthetic, recombinant cDNA expression cassettesfor these molecules were constructed and polypeptides were expressed inCHODG44 cells according to methods described in Example 2.

Purified fusion protein derivatives of CytoxB-scFvlg molecules wereanalyzed by SDS-PAGE according to the methods described in Example 2.Polyacrylamide gels were run under non-reducing and reducing conditions.Two different molecule weight marker sets, BioRad prestained markers,(BioRad, Hercules, Calif.) and Novex Multimark molecular weight markerswere loaded onto each gel. The migration patterns of the differentconstructs and of RituximabTM are presented in FIG. 12.

The ability of the different derivatives of CytoxB-scFvlg molecules tomediate ADCC was measured using the Bjab B lymphoma cells as the targetand freshly prepared human PBMCs as effector cells. (See Example 2.)Effector to target ratios were varied as follows: 70:1, 35:1, and 18:1,with the number of Bjab cells per well remaining constant but the numberof PBMCs were varied. Bjab cells were labeled for 2 hours with ⁵¹Cr andaliquoted at a cell density of 5×10⁴ cells/well to each well offlat-bottom 96 well plates. Purified fusion proteins or rituximab wereadded at a concentration of 10 μg/ml to the various dilutions of PBMCs.Spontaneous release was measured without addition of PBMC or fusionprotein, and maximal release was measured by the addition of detergent(1% NP-40) to the appropriate wells. Reactions were incubated for 4hours, and 100 μl of culture supernatant was harvested to a Lumaplate(Packard Instruments) and allowed to dry overnight prior to counting cpmreleased. The results are presented in FIG. 13.

Complement dependent cytotoxicity (CDC) activity of the CytoxBderivatives was also measured. Reactions were performed essentially asdescribed in Example 2. The results are presented in FIG. 14 as percentof dead cells to total cells for each concentration of fusion protein.

Example 6 In Vivo Studies in Macaques

Initial in vivo studies with CytoxB derivatives have been performed innonhuman primates. FIG. 15 shows data characterizing the serum half-lifeof CytoxB in monkeys. Measurements were performed on serum samplesobtained from two different macaques (J99231 and K99334) after doses of6 mg/kg were administered to each monkey on the days indicated byarrows. For each sample, the level of 2H7scFvIg present was estimated bycomparison to a standard curve generated by binding of purifiedCytoxB-(MHWTG1C)-Ig fusion protein to CD20 CHO cells (see Example 2).The data are tabulated in the bottom panel of the FIG. 15.

The effect of CytoxB-(MHWTG1C)Ig fusion protein on levels of circulatingCD40+ cells in macaques was investigated. Complete blood counts wereperformed at each of the days indicated in FIG. 16. In addition, FACS(fluorescence activated cell sorter) assays were performed on peripheralblood lymphocytes using a CD40-specific fluorescein conjugated antibodyto detect B cells among the cell population. The percentage of positivecells was then used to calculate the number of B cells in the originalsamples. The data are graphed as thousands of B cells per microliter ofblood measured at the days indicated after injection (FIG. 16).

Example 7 Construction and Expression of an Anti-CD 19 SCFV-IG FusionProtein

An anti-CD19 scFv-Ig fusion protein was constructed, transfected intoeukaryotic cells, and expressed according to methods presented inExamples 1, 2, and 5 and standard in the art. The variable heavy chainregions and variable light chain regions were cloned from RNA isolatedfrom hybridoma cells producing antibody HD37, which specifically bindsto CD19. Expression levels of a HD37scFv-IgAHWTG1C and aHD37scFv-IgMHWTG1C were measured and compared to a standard curvegenerated using purified HD37 scFIg. The results are presented in FIG.17.

Example 8 Construction and Expression of an Anti-L6 SCFV-IG FusionProtein

An scFv-Ig fusion protein was constructed using variable regions derivedfrom an anti-carcinoma mAb, L6. The fusion protein was constructed,transfected into eukaryotic cells, and expressed according to methodspresented in Examples 1, 2, and 5 and standard in the art. Expressionlevels of L6scFv-IgAHWTG1C and L6scFv-IgMHWTG1C were measured andcompared to a standard curve generated using purified HD37 scFvIg. Theresults are presented in FIG. 18.

Example 9 Characterization of Various SCFV-IG Fusion Proteins

In addition to the scFv-Ig fusion protein already described, G28-1(anti-CD37) scFv-Ig fusion proteins were prepared essentially asdescribed in Examples 1 and 5. The variable regions of the heavy andlight chains were cloned according to methods known in the art. ADCCactivity of 2H7-MHWTG1C, 2H7-IgAHWTG1C, G28-1-MHWTG1C, G28-1 IgAHWTG1C,HD37- MHWTG1C, and HD37- IgAHWTG1C was determined according to methodsdescribed above (see Example 2). Results are presented in FIG. 19. ADCCactivity of L6scFv-IgAHWTG1C and L6scFv-IgMHWTG1C was measured using the2981 human lung carcinoma cell line. The results are presented in FIG.20. The murine L6 monoclonal antibody is known not to exhibit ADCCactivity.

The purified proteins were analyzed by SDS-PAGE under reducing andnon-reducing conditions. Samples were prepared and gels run essentiallyas described in Examples 2 and 5. The results for the L6 and 2H7 scFv-Igfusion proteins are presented in FIG. 21 and the results for the G28-1and HD37 scFv-Ig fusion proteins are presented in FIG. 22.

Example 10 Construction and Expression of Anti-CD20 SCFV-Llama IG FusionProteins

This Example illustrates the cloning of llama IgG1, IgG2, and IgG3constant region domains and the construction of immunoglobulin fusionproteins with each of the three constant regions and anti-CD20 scFv.

The constant regions of llama IgG1, IgG2, and IgG3 immunoglobulins werecloned and inserted into mammalian vector constructs containing ananti-CD20 single chain Fv, 2H7 scFv. Total RNA was isolated fromperipheral blood mononuclear cells (PBMC) from llama blood (Triple JFarms, Bellingham, Wash.) by lysing the lymphocytes in TRIzol®(Invitrogen Life Technologies, Carlsbad, Calif.) according to themanufacturer's instructions. One microgram (1 μg) of total RNA was usedas template to prepare cDNA by reverse transcription. The RNA and 200 ngrandom primers were combined and denatured at 72° C. for 10 minutesprior to addition of enzyme. Superscript II reverse transcriptase(Invitrogen Life Technologies) was added to the RNA plus primer mixturein a total volume of 25 μl in the presence of 5× second strand bufferand 0.1 M DTT provided with the enzyme. The reverse transcriptionreaction was allowed to proceed at 42° C. for one hour. The cDNA wasamplified by PCR using sequence specific primers. The 5′ primers weredesigned according to published sequences for the V_(HH) and V_(H)domains of camelids. The 3′ primer, which was used to amplify all threeisotypes, was designed using mammalian CH3 domain sequences as a guide.The following specific primers were used. The Bcl and XbaI sites areindicated by underlined italicized sequences.

5′ primer for llama IgG1 constant region (SEQ ID NO: 227) LLG1-5′bgl:5′-gtt gt t gat c aa gaa cca cat gga gga tgc acg tg-3′ 5′ primer forllama IgG2 constant region (SEQ ID NO: 228) LLG2-5′bgl: 5′-gtt gtt gat c aa gaa ccc aag aca cca aaa cc-3′ 5′ primer for llama IgG3constant region (SEQ ID NO: 229) LLG3-5′bgl: 5′-gtt gt t gat c aa gcgcac cac agc gaa gac ccc-3′ 3′ primer for llama IgG1, IgG2, and IgG3constant regions (SEQ ID NO: 238) LLG123-3′X: 5′-gtt gtt tct aga  ttacta ttt acc cga aga ctg ggt gat gga-3′

PCR fragments of the expected size were cloned into TOPO® cloningvectors (Invitrogen Life Technologies) and then were sequenced. Thesense sequencing primer, LLseqsense, had the sequence 5′-ctg aga tcg agttca gct g-3′ (SEQ ID NO: 230), and the antisense primer, LLseqAS, hadthe sequence 5′-cct cct ttg gct ttg tct c-3′ (SEQ ID NO: 231).Sequencing was performed as described in Example 1. FIG. 23 compares theamino acid sequence of the three isotype llama constant regionscontaining the hinge, CH2, and CH3 domains with the amino acid sequenceof human IgG1 hinge, CH2, and CH3 domains.

After verifying the sequence, the amplified PCR products were digestedwith restriction enzymes BclI and Xbal to create compatible restrictionsites. The digested fragments were then gel-purified, and the DNA waseluted using a QlAquick Gel Extraction Kit (QIAGEN, Valencia, Calif.).The 2H7scFv-Ig pD18 mammalian expression vector construct (see Example2) was digested with BclI and XbaI to remove the human IgG hinge, CH2,and CH3 domains. The pD18 vector is a modified derivative of pCDNA3 thatcontains an attenuated DHFR gene, which serves as a selectable markerfor mammalian expression (Hayden et al., Tissue Antigens 48:242-54(1996)). The purified llama IgG1, IgG2, and IgG3 constant region PCRproducts were ligated by T4 DNA ligase (Roche Molecular Biochemicals,Indianapolis, Ind.) into the double-digested 2H7 scFv-pD18 vector atroom temperature overnight according to the manufacturer's instructions.After ligation, the ligation products were transformed into E. coli DH5abacteria (BD Biosciences, Palo Alto, Calif.) and plated according tostandard molecular biology procedures and manufacturer's instructions.Isolated colonies were chosen to screen for transformants containing thecorrect inserts.

For expression of the encoded polypeptides, plasmid DNA from positiveclones was transiently transfected into COS-7 cells using DEAE-dextran(Hayden et al., Ther Immunol. 1:3-15 (1994)). COS-7 cells were seeded atapproximately 3×10⁶ cells per 150 mm plate and grown overnight until thecells were about 75% confluent. Cells were then washed once withserum-free DMEM (Invitrogen Life Technologies, Grand Island, N.Y.).Transfection supernatant (10 ml) containing 400 μg/ml DEAE-dextran, 0.1mM chloroquine, and 5μg/ml of the DNA constructs were added to thecells, which were then incubated at 37° C. for 3-4 hrs. Afterincubation, cells were pulsed with 10 ml of 10% dimethyl sulfoxide(DMSO) in lx PBS at room temperature for 2 minutes. Cells were thenplaced back into fully supplemented DMEM/10% FBS (1% L-glutamine, 1%penicillin/streptomycin, 1% sodium pyruvate, 1% MEM essential aminoacids) (Invitrogen Life Technologies). After 24 hours, the media wasreplaced with serum-free fully supplemented DMEM (Invitrogen LifeTechnologies), and the cells were maintained up to 21 days with mediachanges every 3-4 days.

Ig-fusion proteins were purified by passing COS cell culturesupernatants through Protein A Agarose (Repligen, Cambridge, Mass.)columns. After application of the culture supernatant, the Protein Acolumns were then washed with 1×PBS (Invitrogen Life Technologies).Bound Ig˜fusion proteins were eluted with 0.1 M citric acid (pH 2.8),and the collected fractions were immediately neutralized with Tris base(pH 10.85). The fractions containing protein were identified bymeasuring the optical density (A₂₈₀) and then were pooled, dialyzedagainst 1×PBS, (Invitrogen Life Technologies) and filtered through a 0.2μm filter.

The purified Ig-fusion proteins were analyzed by SDS-PAGE. Aliquots of2H7 scFv-llama IgG1, 2H7 scFv-llama IgG2, 2H7 scFv-llama IgG3, andRituxan® (Rituximab, anti-CD20 antibody, Genentech, Inc. and IDECPharmaceuticals Corp.) (provided by Dr. Oliver W. Press, Fred HutchisonCancer Research Center, Seattle, Wash.) (5 μg proteInd.) were combinedwith 25 μl 2× NuPAGE® SDS Sample Buffer (Invitrogen Life Technologies)(non-reduced samples). Samples of each protein were also prepared inreducing sample buffer containing 5% 2-mercaptoethanol (Sigma-Aldrich,St. Louis, Mo.). Molecular weight markers (Invitrogen Life Technologies)were applied to the gels in non-reducing buffer only. The proteins werefractionated on NuPAGE® 10% Bis-Tris gels (Invitrogen LifeTechnologies). After electrophoresis (approximately 1 hour), the gelswere washed three times, five minutes each, with Distilled Water(Invitrogen Life Technologies) and then stained in 50 ml Bio-SafeCoommassie Stain (BioRad, Hercules, Calif.) overnight at roomtemperature. After a wash in Distilled Water, the gels werephotographed. The migration pattern of each Ig-fusion protein ispresented in FIG. 24.

The ability of the 2H7 scFv-llama Ig fusion proteins to bind to cellsexpressing CD20 was demonstrated by flow cytometry. Serial dilutionsstarting at 25 μg/ml of purified 2H7 scFv-llama IgG1, 2H7 scFv-llamaIgG2, and 2H7 scFv-llama IgG3 were prepared and incubated withCD20-transfected (CD20+) CHO cells (from the laboratory of Dr. S. Skov,Institute of Medical Microbiology and Immunology, Copenhagen Denmark in1% FBS 1×PBS media (Invitrogen Life Technologies) for one hour on ice.After the incubation, the cells were then centrifuged and washed with 1%FBS in 1×PBS. To detect bound 2H7 scFv-llama Ig, the cells wereincubated for one hour on ice with fluorescein-conjugated goatanti-camelid IgG (heavy and light chaInd.) (1:100) (Triple J Farms). Thecells were then centrifuged and resuspended in 1% FBS-1×PBS and analyzedusing a Coulter Epics XL cell sorter (Beckman Coulter, Miami, Fla.). Thedata (percent of maximum brightness) are presented in FIG. 25.

Example 11 Effector Function of Anti-CD20 SCFV-Llama IG Fusion Proteins

This Example demonstrates the ability of anti-CD20 llama IgG1, IgG2, andIgG3 fusion proteins to mediate complement dependent cytotoxicity (CDC)and antibody dependent cell-mediated cytotoxicity (ADCC).

The ability of the 2H7 scFv-llama IgG fusion proteins to kill CD20positive cells in the presence of complement was tested using the BJABhuman B cell line. Rabbit complement was obtained from 3-4 week oldrabbits (Pel-Freez, Brown Deer, Wis.). BJAB cells (2×10⁶ cells/ml) werecombined with rabbit complement (final dilution 1:10) and purified 2H7Ig fusion proteins. 2H7 scFv-llama IgG1, 2H7 scFv-llama IgG2, 2H7scFv-llama IgG3, and 2H7 scFv-human IgG1 wild type hinge-CH2-CH3)(Example 1) were added at 1:3 serial dilutions beginning at aconcentration of 30 μg/ml. After one hour at 37° C., cell viability wasdetermined by counting live and dead cells by trypan blue exclusion(0.4%) (Invitrogen Life Technologies) using a hemacytometer(Bright-line, Horsham, Pa.). The percent killing was calculated bydividing the number of dead cells by the number of total cells(dead+live cells). The data presented in FIG. 26 show that all Ig fusionproteins had CDC activity.

The ADCC activity of the 2H7 scFv-llama IgG fusion proteins wasdetermined using BJAB cells as target cells and human or llamaperipheral blood mononuclear cells (PBMC) as effector cells. BJAB cellswere pre-incubated for approximately 2 hours with ⁵¹Cr (100 μCi)(Amersham Biosciences, Piscataway, N.J.) in fully supplemented IMDM(Invitrogen Life Technologies) containing 15% FBS. The cells were mixedintermittently during the pre-incubation period. Fresh, resting humanPBMC were purified from whole blood using Lymphocyte Separation Media(LSM) (ICN Pharmaceuticals, New York, N.Y.). PBMC were combined withlabeled BJAB cells (5×10⁴ cells per well of 96 well tissue cultureplate) at ratios of 25:1, 50:1, and 100:1. To each combination was added10 μg/ml of purified 2H7 scFv-llama IgG1, 2H7 scFv-llama IgG2, 2H7scFv-llama IgG3, Rituximab, or no anti-CD20 antibody. The mixtures wereincubated for 6 hours at 37° C. Supernatant from each reactioncontaining ⁵¹Cr released from lysed cells was collected onto aLumaPlate-96 filter plate (Packard, Meriden, Conn.), which was driedovernight. The amount of ⁵¹Cr was measured by a TopCount NXT platereader (Packard). FIG. 27 shows that the 2H7 scFv-llama IgG2 fusionprotein was the most effective llama fusion protein in mediating ADCC.Each data point represents the average measurement of triplicate wells.

ADCC activity was affected by the source of effector cells. Llama PBMCwere isolated from llama blood (Triple J Farms) using LSM. Llamaeffector cells were added at the same ratios to BJAB target cells asdescribed for the ADCC assay using human effector cells. The cells werecombined with 10 μg/ml of purified 2H7 scFv-llama IgG1, 2H7 scFv-llamaIgG2, 2H7 scFv-llama IgG3, Rituximab, or no anti-CD20 antibody. Theresults are presented in FIG. 28.

Example 12 Construction and Characterization of SCFV IG Fusion ProteinsExpressed on the Cell Surface

This Example describes a retroviral transfection system for ectopicsurface expression of genetically engineered cell surface receptorscomposed of scFvs that bind costimulatory receptors. The Example alsodemonstrates the effector function of these various scFv Ig fusionproteins expressed on the surface of target cells.

The heavy and light chain variable regions were cloned from murinemonoclonal antibodies specific for various costimulatory receptors, andsingle chain Fv constructs were prepared essentially as described inExample 1. Antibodies included 2H7, anti-human CD20; 40.2.220,anti-human CD40; 2E12, anti-human CD28; 10A8, anti-human CD152(anti-CTLA-4); and 500A2, anti-murine CD3. The heavy chain and lightchain variable regions of each antibody were cloned according tostandard methods for cloning immunoglobulin genes and as described inExample 1. Single chain Fv constructs were prepared as described inExample 1 by inserting a nucleotide sequence encoding a (gly4ser)₃peptide linker between the VL region nucleotide sequence of 40.2.220,2E12, 10A8, and 500A2, respectively (SEQ ID NOs: 243 or 462;249 or468;474; 263, respectively) and the VH region nucleotide sequence of40.2.220, 2E12, 10A8, and 500A2, respectively (SEQ ID NOs: 245 or 464;251 or 470; 261 or 476; 257, respectively). The polypeptide sequence forVL of 40.2.220, 2E12, 10A8, and 500A2 are set forth in SEQ ID NOs: 244or 463; 250 or 469;255 or 475; 264, respectively, and the polypeptidesequence for VH of 40.2.220, 2E12, 10A8, and 500A2 are set forth in SEQID NOs: 246 or 465; 252 or 471; 258 or 477; 262, respectively. Each scFvpolynucleotide (SEQ ID NOs: 247 or 466;253 or 472;259 or 478;399 for40.2.220, 2E12, 10A8, and 500A2, respectively) was then fused to humanIgG1 mutant hinge (CCC→SSS) and mutant CH2 (proline to serine mutationat residue 238 (238 numbering according to EU nomenclature, Ward et al.,1995 Therap. Immunol. 2:77-94; residue 251 according to Kabat et al.)and wild type CH3 domains according to the methods described in Example5 and 11. Each scFv mutant IgG1 fusion polynucleotide sequence was thenfused in frame to sequences encoding the transmembrane domain andcytoplasmic tail of human CD80 (SEQ ID NO: 460), such that when thefusion protein was expressed in the transfected cell, CD80 provided ananchor for surface expression of the scFv Ig fusion protein. cDNAsencoding the scFv-IgG-CD80 fusion proteins (SEQ ID NOs: 268 or 483; 265or 481; 270 or 485; 272 or 487 for 40.2.220-, 2E12-, 10A8-, and500A2-scFv-IgG-CD80, respectively) were inserted into the retroviralvector pLNCX (BD Biosciences Clontech, Palo Alto, Calif.) according tostandard molecular biology procedures and vendor instructions. ThescFv-Ig-CD80 cDNA was inserted between the 5′LTR-neomycin resistancegene-CMV promoter sequences and the 3′LTR sequence. The retroviralconstructs were transfected into Reh, an acute lymphocytic leukemia cellline (ATCC CRL-8286). Transfected cells were screened to select clonesthat were expressing scFv-Ig fusion proteins on the cell surface.

CDC and ADCC assays were performed with the transfected Reh cells todetermine if expression of the scFv-Ig polypeptides on the cell surfaceaugmented effector cell function. Reh cells expressing anti-human CD152scFv-mutant IgG-CD80 (SEQ ID NO: 270 or 485); Reh anti-human CD28 scFv-mutant IgG-CD80 (SEQ ID NOS: 268 or 483); Reh anti-human CD40 scFv-mutant IgG-CD80 (SEQ ID NOS: 265 or 481); Reh anti-human CD20 scFv-mutant IgG-CD80 were combined with human PBMC (see Example 11) andrabbit complement (10 μml) for one hour at 37° C. Untransfected Rehcells were included as a control. Viability of the cells was determinedby trypan blue exclusion, and the percent of killed cells was calculated(see Example 11). FIG. 29 shows the effectiveness of the scFv-IgG-CD80fusion proteins when expressed on the cell surface of tumor cells tomediate complement dependent cytotoxicity.

The same transfected Reh cells tested in the CDC assay plus Reh cellstransfected with the polynucleotide construct that encodes anti-murineCD3-scFv-Ig-CD80 (SEQ ID NO: 486) were analyzed for ADCC activity (seeExample 11). Untransfected and transfected Reh cells were pre-labeledwith ⁵¹Cr (100 μCi) (Amersham) for two hours at 37° C. Human PBMC servedas effector cells and were added to the Reh target cells (5×10⁴ cellsper well of 96 well plate) at ratios of 5:1, 2.5:1, and 1.25:1. Afterfive hours at 37° C., culture supernatants were harvested and analyzedas described in Example 11. Percent specific killing was calculatedaccording to the following equation: ((experiment release minusspontaneous release)/(maximum release minus spontaneous release))×100.The data are presented in FIG. 30. Each data point represents theaverage of quadruplicate samples.

Using the same procedures described above, the same results with otherbinding domains were obtained using the following monoclonal antibodiesmAbs as sources of sFv: for CD20, 1F5 (Genbank AY 058907 and AY058906);for CD40, 2.36 and G28.5; for CD28, 9.3.

Cell surface expression of antibody binding domains is accomplished byfusing antibody scFvs to IgA hinge and constant regions and IgE hingeand constant regions. Polynucleotides encoding an anti-4-1BB scFv, 5B9(anti-human 4-1BB) scFv, and 2e12 (anti-human CD40) fused to IgAH IgA T4(four terminal CH3 residues deleted) fused to the CD80 transmembrane andcytoplasmic domains and IgE Fc regions are shown in SEQ ID NOs: 626 and630. The encoded polypeptides are shown in SEQ ID NOs: 627 and 631.

Example 13 Construction and Sequence of Human IG HinGE-CH2-CH3 Mutantsand 2H7 Variable Region Mutants

This Example describes construction of scFv fusion proteins containingmutant human IgG1 and IgA constant regions. This Example also describesconstruction of a 2H7 scFv mutant with a single point mutation in thevariable heavy chain region. Mutations were introduced into variable andconstant region domains according to methods described herein and knownin the molecular biology arts. FIG. 31 presents nomenclature for the Igconstant region constructs.

The human IgG1 hinge region of the 2H7 scFv human IgG1 hinge-CH2-CH3fusion proteins was mutated to substitute cysteine residues that in awhole immunoglobulin are involved in forming disulfide bonds between twoheavy chain molecules. One mutant, 2H7 scFv fused to a human IgG1 hingeregion in which all three cysteine residues were mutated to serineresidues (MTH (SSS)), was prepared as described in Example 5 (designatedin Example 5 as CytoxB-MHWTG1C (includes wild type IgG1 CH2 and CH3domains)) (now referred to as 2H7 scFv MTH (SSS) WTCH2CH3) and comprisesthe polynucleotide sequence SEQ ID NO: 4 encoding the polypeptide as setforth in SEQ ID NO: 17. The polynucleotide sequence encoding this mutant(SEQ ID NO: 4) was used as a template to create mutant hinge regions inwhich the first two cysteine residues were substituted with serineresidues (IgG MTH (SSC)). An oligonucleotide was designed to substitutethe third serine residue with a cysteine and had the following sequence:5′-gtt gtt gat cag gag ccc aaa tct tct gac aaa act cac aca tct cca ccgtgc cca gca cct g-3′ (HuIgGMHncs3, SEQ ID NO: 275). A second mutant wasprepared in which the mutant hinge had serine residues substituting thefirst and third cysteine residues (IgG MTH (SCS)). The sequence of theoligonucleotide to create this mutant was as follows: 5′-gtt gtt gat caggag ccc aaa tct tct gac aaa act cac aca tgc cca ccg-3′ (HuIgGMHncs2, SEQ

ID NO: 276). A third mutant was prepared with cysteine residuessubstituted at the second and third positions (IgG MTH (CSS)), alsousing the IgG MTH (SSS) mutant as template, and an oligonucleotidehaving the sequence, 5′-gtt gtt gat cag gag ccc aaa tct tgt gac aaa actcac-3′ (HuIgGMHncsl, SEQ ID NO: 277).

The oligonucleotides introducing the mutations into the hinge regionwere combined with template and a 3′ oligonucleotide containing an XbaIsite (underlined and italicized) (5′-gtt gtt tct aga tca ttt acc cgg agacag gga gag get ctt ctg cgt gta g-3′ (SEQ ID NO: 278)) to amplify themutant hinge-wild type (WT)-CH2-CH3 sequences by PCR. The IgG MTH CSSand IgG MTH SCS mutant sequences were amplified for 25 cycles with adenaturation profile of 94° C., annealing at 52° C. for 30 seconds, andextension at 72° C. for 30 seconds. The IgG MTH SSC mutant sequence wasamplified under slightly different conditions: denaturation profile of94° C., annealing at 45° C. for 30 seconds, and extension at 72° C. for45 seconds. The amplified polynucleotides were inserted into the TOPO®cloning vector (Invitrogen Life Technologies) and then were sequenced asdescribed in Example 1 to confirm the presence of the mutation. pD18vector containing 2H7 scFv was digested to remove the constant regionsequences essentially as described in Example 10. The mutant hinge-wildtype CH2-CH3 regions were inserted in frame into the digested vector DNAto obtain vectors comprising 2H7 scFv MTH (CSS) WTCH2CH3 encoding DNA(SEQ ID NO: 581); 2H7 scFv MTH (SCS) WTCH2CH3 encoding DNA (SEQ ID NO:583); and 2H7 scFv MTH (SSC) WTCH2CH3 encoding DNA (SEQ ID NO: 585).

A mutation of leucine to serine at position 11 in the first frameworkregion of the heavy chain variable region (numbering according to Kabatet al., Sequences of Proteins of Immunological Interest, 5^(th) ed.Bethesda, Md.: Public Health Service, National Institutes of Health(1991)) was introduced into the 2H7 scFv MTH (SSS) WTCH2CH3 fusionprotein (SEQ ID NOS: 4 or 488). The wild type leucine residue wassubstituted with serine by site-directed mutagenesis using theoligonucleotide Vhserll: 5′-gga ggt ggg agc tct cag gct tat cta cag cagtct ggg gct gag tcg gtg agg cc-3′ (SEQ ID NO: 279). The 3′-primer forPCR was huIgGl-3′ having the sequence 5′-gtc tct aga cta tca ttt acc cggaga cag-3′ (SEQ ID NO: 280) (XbaI site underlined and italicized). AfterPCR amplification, the fragments were inserted into the TOPO® cloningvector and sequenced to confirm the presence of the VH11 leucine toserine mutation. The 2H7 scFv-IgG MTH (SSS) WTCH2CH3 encoding DNA wasshuttled into the PSL1180 cloning vector (Pharmacia Biotech, Inc.,Piscataway, N.J.). The construct PSL1180-2H7 scFv-IgG MTH (SSS) WTCH2CH3was digested with Sac and XbaI to remove the wild type VH domain and thehinge and CH2 and CH3 domains. The PCR product comprising the VH11mutant was digested with Sac and XbaI and then inserted into thedigested PSL1180 construct according to standard molecular biologyprocedures. The construct was then digested with Hind III and XbaI, andinserted into the mammalian expression vector pD18 (see methodsdescribed in Example 1 and Example 10). The mutant is designated 2H7scFv VH11SER IgG MTH (SSS) WTCH2CH3 (FIG. 31). Four constructscontaining IgA constant region domains were prepared. One constructcontained wild type IgA hinge fused to human IgG1 CH2 and CH3 (IgAH IgGWTCH2CH3) (FIG. 31). Sequential PCR amplifications were performed tosubstitute the human IgG1 hinge of the 2H7 scFv construct withnucleotide sequences encoding the IgA hinge. The 5′ oligonucleotideprimer (huIgA/Gchim5) for the first PCR reaction had the sequence,5′-cca tct ccc tca act cca cct acc cca tct ccc tca tgc gca cct gaa ctcctg-3′ (SEQ ID NO: 281). The primer (huIgAhg-5′) for the second PCRreaction to add more IgA specific hinge sequence and add a BclIrestriction enzyme site (italicized and underlined) had the sequence,5′-gtt gtt gat cag cca gtt ccc tca act cca cct acc cca tct ccc caa ct-3′(SEQ ID NO: 282). The 3′ primer for both amplification steps washuIgGl-3′ having the sequence, 5′-gtc tct aga cta tca ttt acc cgg agacag-3′ (SEQ ID NO: 280). The sequence of the PCR product was confirmedby TOPO® cloning as described above. The gel-purified fragment wasdigested with BclI and XbaI and then inserted into the 2H7 scFv-pD18vector that had been digested BclI and XbaI to remove all the IgG1constant region domains. Ligation was performed as described in Example10 to provide a mammalian expression vector comprising the nucleotidesequence (SEQ ID NO: 283) encoding a 2H7 scFv IgA hinge-IgG1 CH2-CH3polypeptide (SEQ ID NO: 284).

A second pD18 mammalian expression vector was constructed that had apolynucleotide sequence (SEQ ID NO: 1) that encoded a 2H7 scFv fused towild type IgA hinge, CH2, and CH3 domains (SEQ ID NO: 285). Human IgAconstant regions sequences were obtained by using random primers toreverse transcribe total RNA isolated from human tonsil followed by PCRamplification of the cDNA using sequence specific primers, essentiallyas described in Example 10. Human IgA hinge-CH2-CH3 nucleotide sequence(SEQ ID NO: 285) encoding the IgA-CH2-CH3 polypeptide (IgAH IgACH2CH3,FIG. 31) (SEQ ID NO: 286) was amplified using the 5′ oligonucleotidehuIgAhg-5′ (SEQ ID NO:(same as above, SEQ ID NO: 281) and a 3′oligonucleotide huIgA3′ having the sequence, 5′-gtt gtt tct aga tta tcagta gca ggt gcc gtc cac ctc cgc cat gac aac-3′ (SEQ ID NO: 289).Secretion of a 2H7-IgA hinge-IgA CH2-CH3 polypeptide from transfectedmammalian cells required co-expression of human J chain that covalentlybinds to two IgA CH3 domains via disulfide bonds. Total RNA was isolatedfrom tonsil B cells and was reversed transcribed to generate cDNA asdescribed above. PCR amplification of the nucleotide sequence encodingthe J chain was performed with J chain specific primers. The 5′ PCRprimer, HUJCH5nl, had the sequence, 5′-gtt gtt aga tct caa gaa gat gaaagg att gtt ctt-3′ (SEQ ID NO: 292), and sequence of the 3′ primer,HUJCH3, was 5′-gtt gtt tct aga tta gtc agg ata gca ggc atc tgg-3′ (SEQID NO: 293). The cDNA was cloned into TOPO® for sequencing as describedin Example 10. J chain encoding cDNA (SEQ ID NO: 290) was then insertedinto pD18 and pCDNA3-Hygro (+) (Invitrogen Life Technology) vectors forco-transfection with 2H7 scFv IgA hinge-CH2-CH3 constructs. The J chainhas the predicted amino acid sequence set forth in SEQ ID NO: 291.

Secretion of an scFv IgA constant region construct in the absence of Jchain was accomplished by engineering a truncated CH3 domain with adeletion of the four carboxy terminal amino acids (GTCY, SEQ ID NO: 294)(IgAH IgA-T4, FIG. 31), which include a cysteine residue that forms adisulfide bond with the J chain. The IgA hinge-CH2-CH3 nucleotidesequence containing the deletion in CH3 (SEQ ID NO: 295) was preparedusing a 5′ PCR primer (huIgAhg-5′) having the sequence 5′-gtt gtt gatcag cca gtt ccc tca act cca cct acc cca tct ccc tca act-3′ (SEQ ID NO:310) (BclI site is underlined and italicized), and a 3′ PCR primer(HUIGA3T1) having the sequence 5′-gtt gtt tct aga tta tca gtc cac ctccgc cat gac aac aga cac-3′ (SEQ ID NO: 297). This mutated IgA constantregion nucleotide sequence was inserted into a 2H7 scFv pD18 vector asdescribed for the generation of the previous 2H7 scFv-Ig constructs (seeExample 1 and this example) that comprises the polynucleotide sequence(SEQ ID NO: 298) encoding a 2H7 IgAH IgA-T4 polypeptide (SEQ ID NO:299).

A fourth construct was prepared that encoded a 2H7 scFv-IgA constantregion fusion protein with a deletion of 14 additional amino acids, mostof which are hydrophobic residues, from the carboxy terminus of IgA CH3.The 2H7 scFv-IgAH IgA-T4 encoding polynucleotide was used as template toengineer a deletion of the nucleotide sequence encoding PTHVNVSVVMAEVD(SEQ ID NO: 300). The 5′ oligonucleotide primer had the sequence 5′-gttgtt gat cag cca gtt ccc tca act cca cct acc cca tct ccc tca act-3′ (SEQID NO: 310) (BclI site shown as underlined and italicized). The 3′oligonucleotide sequence was 5′-gtt gtt tct aga tta tca ttt acc cgc caagcg gtc gat ggt ctt-3′ (SEQ ID NO: 301). The deleted IgA CH3 region wasamplified by using the above oligonucleotides to amplify the IgAconstant region from RNA isolated from human tonsil such that the cDNAcontained the deleted carboxyl terminal encoding region for the 18 aminoacids. The IgAH IgA-T18 constant region was inserted into a 2H7 scFvpD18 vector that comprises the polynucleotide sequence (SEQ ID NO: 302)encoding a 2H7 IgAH IgA-T18 polynucleotide (SEQ ID NO: 303) as describedabove.

Example 14 Effector Function of CTLA-4 IGG Fusion Proteins

The Example compares the effector functions of CTLA-4 Ig fusion proteinsin CDC and ADCC assays.

Two CTLA-4 IgG fusion proteins were constructed. One fusion proteincomprises the extracellular domain of CTLA-4 fused to human IgG1 wildtype hinge, CH2, and CH3 domains and is designated CTLA-4 IgG WTH (CCC)WTCH2CH3 (SEQ ID NO: 307). A pD18 mammalian expression vector comprisinga polynucleotide sequence encoding CTLA-4 IgG WTH (CCC) WTCH2CH3 (SEQ IDNO: 306) was prepared by fusing in frame the nucleotide sequenceencoding the extracellular domain of CTLA-4 (SEQ ID NO: 308) (see U.S.Pat. No. 5,844,095) to the nucleotide sequence encoding IgG WTH (CCC)WTCH2CH3 according to the methods described in Examples 1 and 10. Theextracellular domain nucleotide sequence also comprises a BclIrestriction enzyme site at the 3′ end, and a leader peptide nucleotidesequence (SEQ ID NO: 313) that encodes an oncoM leader peptide (SEQ IDNO: 314). A second CTLA-4 IgG fusion protein, designated CTLA-4 IgG MTH(SSS) MTCH2WTCH3, contained the extracellular domain of CTLA-4 (plus theoncoM leader peptide sequence) fused to a mutant IgG hinge in which allthree cysteine residues were replaced with serine residues. The hingeregion was fused to a mutant IgG1 CH2 domain that had a mutation atisotype position 238 (EU numbering, Ward et al., supra, (position 251using numbering according to Kabat et al., supra; position 209 wherenumbering commences with first residue of IgG1 CH1; i.e., PAPELLGGPS(SEQ ID NO: 537) of wild type IgG1 CH2 is modified to PAPELLGGSS (SEQ IDNO: 583), which was fused to IgG1 wild type CH3 (U.S. Pat. No.5,844,095). The CTLA-4 IgG MTH (SSS) MTCH2WTCH3 polynucleotide comprisesthe nucleotide sequence in SEQ ID NO: 315 and the deduced amino acidsequence comprises the sequence provided in SEQ ID NO: 316. CTLA-4fusion proteins were also prepared using CTLA-4 extracellular membraneencoding sequences without the leader peptide (SEQ ID NO: 313 (DNA) and314 (AA)).

To measure CDC activity, purified CTLA-4 IgG WTH (CCC) WTCH2CH3 (2 μml)or CTLA-4 IgG MTH (SSS) MTCH2WTCH3 (2 μg/ml) was added to Reh cells (seeExample 12) and to Reh cells transfected with the costimulatory moleculeCD80 such that CD80 was expressed on the cell surface (Reh CD80.10,obtained from Dr. E. Clark, University of Washington, Seattle, Wash.;see Doty et al., 1998 J. Immunol. 161:2700; Doty et al., 1996 J.Immunol. 157:3270), in the presence or absence of rabbit complement (10μg/ml). Purified CTLA Ig fusion proteins were prepared from culturesupernatants of transiently transfected COS cells according to methodsdescribed in Example 10. The assays were performed essentially asdescribed in Example 11 and 12. The data presented in FIG. 32 show thatonly CD80-transfected Reh cells were killed in the presence ofcomplement and CTLA-4 IgG WTH (CCC) WTCH2CH3 fusion protein.

The purified CTLA-4 Ig fusion proteins were also tested in ADCC assays.Human PBMC, serving as effector cells, were added to Reh or Reh CD80.1target cells at a ratio of 1.25:1, 2.5:1, 5.0:1, and 10:1. Cells werelabeled and the assays performed essentially as described in Examples 11and 12. The results are presented in FIG. 33. Each data point representsthe average of four independent culture wells at each effector:targetcell ratio. The data show that only CTLA-4 IgG WTH (CCC) WTCH2CH3mediated significant ADCC of Reh CD80.10 cells.

Example 15 Effector Function of CTLA-4 IGA Fusion Proteins

CTLA-4 IgA fusion proteins are prepared as described for the IgG fusionproteins (see Examples 1, 13, and 14). CTLA-4 extracellular domainnucleotide sequence (SEQ ID NO: 313) is fused in open reading frame tonucleotides encoding IgAH IgACH2CH3 (SEQ ID NO: 287) to provide thenucleotide sequence (SEQ ID NO: 319) encoding a CTLA-4 IgAH IgACH2CH3fusion protein (SEQ ID NO: 320). The fusion protein is transientlyexpressed in COS cells (see Example 10) or stably expressed in CHO cells(see Example 1). Secretion of the CTLA-4 IgAH IgACH2CH3 fusion proteinrequires co-transfection with a construct containing a polynucleotidesequence (SEQ ID NO: 290) that encodes human J chain (SEQ ID NO: 291).The CTLA-4 IgAH IgACH2CH3 fusion protein is isolated as described inExamples 10 and 14. To express a CTLA-4 IgA construct without thepresence of J chain, a CTLA-4 IgAH IgA-T4 construct is prepared andtransfected into mammalian cells. In a similar manner as described forthe CTLA-4 extracellular fragment fused to wild type IgA hinge-CH2CH3,the CTLA-4 extracellular domain nucleotide sequence (SEQ ID NO: 308) isfused in open reading frame to a nucleotide sequence (SEQ ID NO: 295)encoding a IgAH IgA-T4 polypeptide (SEQ ID NO: 296) to provide anucleotide sequence comprising SEQ ID NO: 319 encoding a CTLA-4 IgAHIgA-T4 polypeptide (SEQ ID NO: 320). Effector function of each constructis evaluated by CDC and ADCC as described in Example 14.

Example 16 Binding of Anti-CD20 SCFV Human IG Fusion Proteins to CHOCells Expressing CD20

This Example describes binding of 2H7 scFv Ig fusion proteins to CHOcells that express CD20. The analysis was performed by flow cytometry.Culture supernatants were collected from transiently transfected COScells expressing 2H7 scFv IgG WTH (CCC) WTCH2CH3 (SEQ ID NOS: 2, 15;239, 240; 458, 459); 2H7 scFv IgG MTH (CSS) WTCH2CH3 (SEQ ID NO: 582);2H7 scFv IgG MTH (SCS) WTCH2CH3 (SEQ ID NO: 584); and 2H7 scFv VHSER11WTH WTCH2CH3, and two-fold serial dilutions were prepared. Serialtwo-fold dilutions of purified 2H7 scFv IgG MTH (SSC) WTCH2CH3 (SEQ IDNOS: 585, 586) were prepared starting at a concentration of 5 μg/ml. Theculture supernatants and purified fusion protein samples were incubatedwith (CD20+) CHO cells for one hour on ice. The cells were washed twiceand then incubated with 1:100 FITC-conjugated goat anti-human IgG(CalTag) for 40 minutes. The unbound conjugate was then removed bywashing the cells and flow cytometry analysis was performed using aCoulter Epics XL cell sorter. Results are presented in FIG. 34.

Example 17 Immunoblot Analysis of Anti-CD20 SCFV Human IGG and IGAFusion Proteins

This Example describes immunoblot analysis of 2H7 scFv IgG and 2H7 scFvIgA fusion proteins that were immunoprecipitated from transfected cellculture supernants.

COS cells were transiently transfected with plasmids comprisingnucleotide sequences for 2H7 scFv IgG WTH (CCC) WTCH2CH3 (SEQ ID NO:458); 2H7 scFv IgG MTH (CSS) WTCH2CH3 (SEQ ID NO: 581); 2H7 scFv IgG MTH(SCS) WTCH2CH3 (SEQ ID NO: 583); 2H7 scFv IgA H IgG WTCH2CH3 (SEQ IDNOS: 283, 499); and scFv IgG MTH (SSS) WTCH2CH3 (SEQ ID NOS: 273, 488)essentially according to the method described in Example 10. Cells werealso transfected with vector only. After 48-72 hours at 37° C., cellculture supernatants were harvested and combined with protein A-agarosebeads (Repligen) for one hour at 4° C. The beads were centrifuged andwashed several times in TNEN [20 mM Tris base, 100 mM NaCl, mM EDTA, and0.05% NP-40, pH 8.0). The immunoprecipitates were combined with 25 μl 2×NuPAGE® SDS Sample Buffer (Invitrogen Life Technologies) (non-reducedsamples). The proteins were fractionated on NuPAGE® 10% Bis-Tris gels(Invitrogen Life Technologies). After electrophoresis (approximately 1hour), the proteins were transferred from the gel onto a Immobilon Ppolyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, Mass.)using a semi-dry blotter (Ellard Instrumentation, Monroe, Wash.). ThePVDF membrane was blocked in PBS containing 5% nonfat milk and thenprobed with HRP-conjugated goat anti-human IgG (Fc specific) (CalTag).After washing the immunoblot several times in PBS, the blot wasdeveloped using ECL (Amersham Biosciences). The results are shown inFIG. 35.

Example 18 Binding of Anti-CD20 SCFV Human IGA Fusion Proteins toCD20+CHO Cells

This Example describes flow immunocytofluorimetry analysis of binding of2H7 scFv IgAH IgACH2CH3 (SEQ ID NOS: 286, 502) and 2H7 scFv IgAH IgAT4(SEQ ID NOS: 299, 515) fusion proteins to (CD20+) CHO cells.

COS cells were transiently co-transfected as described in Example 10with plasmid DNA comprising a polynucleotide sequence (SEQ ID NO: 285)encoding 2H7 scFv IgAH IgACH2CH3 polypeptide (SEQ ID NO: 501) and with aseparate plasmid comprising a polynucleotide sequence (SEQ ID NO: 290)encoding a human J chain polypeptide (SEQ ID NO: 291). COS cells werealso transfected with a polynucleotide sequence (SEQ ID NOS: 298, 514)encoding a 2H7 scFv IgA fusion protein that had a deletion of four aminoacids at the carboxy terminus of CH3 (2H7 scFv IgAH IgA-T4, SEQ ID NOS:299, 515). The transfections were performed as described in Example 10.Culture supernatants from transfected COS cells were combined with(CD20+) CHO cells (see Example 1) and incubated for one hour on ice. Thecells were washed twice with PBS-2% FBS and then combined withFITC-conjugated goat anti-human IgA chain (CalTag) (1:100) for 40minutes. The cells were again washed and then analyzed by flow cytometryusing a Coulter Epics XL cell sorter. FIG. 36 shows that co-transfectionwith J chain was not required for secretion of 2H7 scFv IgAH IgAT4, the2H7 IgA fusion protein with the truncated CH3 carboxy end (SEQ ID NO:299, 515).

Example 19 Effector Function of Anti-CD20 SCFV Human IGA Fusion Proteins

This Example illustrates ADCC activity of 2H7 IgG and IgA fusionproteins against cells that express CD20. BJAB cells were prelabeledwith ⁵¹Cr (100 μCi) (Amersham) for two hours at 37° C. Effector cellswere obtained from fresh, resting human whole blood, which was dilutedin an equal volume of Alsever's solution to prevent coagulation. 2H7scFv IgG MTH (SSS) WTCH2CH3 (SEQ ID NO: 489); 2H7 scFv IgG MTH (SCS)WTCH2CH3 (SEQ ID NO: 586); 2H7 scFv IgG WTH (CCC) WTCH2CH3 (SEQ ID NO:459); and 2H7 scFv IgAH IgACH2CH3 (SEQ ID NO: 299, 515) fusion proteinswere purified from transiently transfected COS cell supernatants(100-200 ml) by protein A chromatography as described in Example 10. COScells transfected with the plasmid encoding 2H7 scFv IgAH IgACH2CH3 wereco-transfected with a plasmid encoding human J chain as described inExample 18. Two-fold serial dilutions of the purified 2H7 Ig fusionproteins starting at 5μg/ml were added to the labeled BJAB cells (5×10⁴cells per well of 96 well tissue culture plate) in the presence of wholeblood (100 μl of whole blood diluted 1:1 in Alsever's solution, finaldilution 1:4) and incubated for five hours at 37° C. Culturesupernatants were harvested and analyzed as described in Example 11.Percent specific killing was calculated according to the followingequation: ((experiment release minus spontaneous release)/(maximumrelease minus spontaneous release))×100. The data are presented in FIG.37. Each data point represents the average of quadruplicate samples.

In a second ADCC assay, the number of labeled BJAB target cells was heldconstant in each sample, and whole blood was added at dilutions of 0.25,0.125, and 0.0625. Purified 2H7 IgG and IgA fusion proteins were addedat a concentration of 5 μg/ml. The BJAB cells, whole blood, and fusionproteins were incubated, the supernatants harvested, and the percentspecific killing was calculated as described above. Percent specifickilling for each of the 2H7 fusion proteins is presented in FIG. 38.

The ADCC activity of purified 2H7 scFv IgG MTH (SSS) WTCH2CH3 (5 μg/ml)and of purified 2H7 scFv IgAH IgACH2CH3 (5 μg/ml) was compared in thepresence of different effector cell populations. PBMC were isolated fromwhole blood as described in Examples 11 and 12. PBMC were combined withlabeled BJAB target cells (5×10⁴ per well of 96 well tissue cultureplate) at ratios of 50:1, 25:1, and 12.5:1. The assay was performed andthe data analyzed as described above. FIG. 39A shows that only the 2H7scFv IgG MTH (SSS) WTCH2CH3 fusion protein had ADCC activity when PBMCserved as the effector cells. FIG. 39B shows that both 2H7 scFv IgG MTH(SSS) WTCH2CH3 and 2H7 scFv IgAH IgACH2CH3 exhibit ADCC activity whenwhole blood was the source of effector cells (as illustrated in FIG.38).

Example 20 Expression Level of 2H7 SCFV VH11 SER IGG MTH (SSS) WTCH2CH3Fusion Protein

This Example compares the expression level of 2H7 scFv VH11Ser IgG MTH(SSS) WTCH2CH3 fusion protein (SEQ ID NO: 488) with other 2H7 scFv IgGconstructs that do not contain the mutation in the variable heavy chaindomain. The mammalian expression vector pD 18 comprising nucleotidesequences 2H7 scFv IgG MTH (SSS) WTCH2CH3 (SEQ ID NO: 488); 2H7 scFv IgGMTH (CSS) WTCH2CH3 (SEQ ID NO: 581); 2H7 scFv IgG MTH (SCS) WTCH2CH3(SEQ ID NO: 583); 2H7 scFv IgG WTH (CCC) WTCH2CH3 (SEQ ID NO: 458); and2H7 scFv VHSER11 IgG MTH (SSS) WTCH2CH3 (see Examples 1 and 13) weretransiently transfected into COS cells as described in Example 10. After72 hours at 37° C., culture supernatants were harvested, and 1 μl ofeach supernatant was combined with non-reducing sample buffer (seemethod described in Example 10). The culture supernatant samples andaliquots of purified 2H7 scFv IgG MTH (SSS) WTCH2CH3 (40 ng, 20 ng, 10ng/5 ng, and 2.5 ng) were fractionated on 10% Bis-Tris (MOPS) NuPAGE®gels (Invitrogen Life Technologies). Multimark® protein standards(Invitrogen Life Technologies) were also separated on the gel. Theproteins were transferred to a PDVF membrane and immunoblotted asdescribed in Example 17. The immunoblot is presented in FIG. 40. Theamounts of the fusion proteins were quantified by densitometry analysisof the blots using the Scionlmage for Windows software and comparisonwith the standard curve. The 2H7 scFv IgG WTH (CCC) WTCH2CH3 constructproduced approximately 12 ng/ul or 12 micrograms/ml, the 2H7 scFv IgGMTH (CSS) WTCH2CH3 produced approximately 10 ng/ul or 10 micrograms/ml,the 2H7 scFv IgG MTH (SCS) WTCH2CH3 construct produced approximately 1ng/ul or 1 microgram/ml, and the 2H7 scFv VHSER11 IgG MTH (SSS) WTCH2CH3construct produced approximately 30 ng/ml or 30 micrograms/ml.

Example 21 Construction of A 2H7 SCFV IGG Fusion Protein with A MutantCH3 Domain

Amino acid mutations were introduced into the CH3 domain of a 2H7 IgGfusion protein. The pD18 vector comprising 2H7 scFv IgG MTH (SSS)WTCH2CH3 (SEQ ID NO: 488) was digested with BclI and XbaI to remove theMTH WTCH2CH3 (SEQ ID NO: 6) fragment, which was then subcloned intopShuttle vector (BD Biosciences Clontech, Palo Alto, Calif.) that wasdouble-digested with BclI and XbaI. Subcloning was performed in akanamycin resistant vector because the ampicillin resistance gene has anXmnI site, which is required for this cloning procedure. Five constructswere prepared with the following substitutions: (1) a phenylalanineresidue at position 405 (numbering according to Kabat et al. supra) wassubstituted with tyrosine using the oligonucleotide CH3Y405; (2) thephenylalanine position at 405 was substituted with an alanine residueusing the oligonucleotide CH3A405; (3) the tyrosine residue at position407 was substituted with an alanine using the oligonucleotide CH3A407;(4) both wild type amino acids at positions 405 and 407 were substitutedwith tyrosine and alanine, respectively using the oligonucleotideCH3Y405A407; and (5) both wild type amino acids at positions 405 and 407were substituted with alanine using the oligonucleotide CH3A405A407. Theoligonucleotides were the 3′ primers for PCR amplification of a portionof the CH3 domain. The nucleotide sequences for each 3′ oligonucleotidewere as follows.

(SEQ ID NO: 365) CH3Y405: 5′-gtt gtt gaa gac gtt ccc ctg ctg cca cct gctctt gtc cac ggt gag ctt gct gta gag gta gaa gga gcc-3′ (SEQ ID NO: 366)CH3A405: 5′-gtt gtt gaa gac gtt ccc ctg ctg cca cct gct ctt gtc cac ggtgag ctt gct gta gag ggc gaa gga gcc-3′ (SEQ ID NO: 367) CH3A407: 5′-gttgtt gaa gac gtt ccc ctg ctg cca cct gct ctt gtc cac ggt gag ctt gct ggcgag gaa gaa gga gcc-3′ (SEQ ID NO: 368) CH3Y405A407: 5′-gtt gtt gaa gacgtt ccc ctg ctg cca cct gct ctt gtc cac ggt gag ctt gct ggc gag gta gaagga gcc-3′ (SEQ ID NO: 369) CH3A405A407: 5′-gtt gtt gaa gac gtt ccc ctgctg cca cct gct ctt gtc cac ggt gag ctt gct ggc gag ggc gaa gga gcc-3′

The template was the mutant hinge MHWTCH2CH3 human IgGl. The 5′ PCRoligonucleotide primer was huIgGMHWC, (SEQ ID NO: 332). The amplifiedproducts were TOPO® cloned and sequenced as described in Examples 1 and10. DNA from the clones with the correct sequence was digested with BclIand XmnI and transferred to pShuttle containing the MTH WTCH2CH3sequence, which was also digested with the same restriction enzymes. Themutated IgG sequences were then removed by digestion with BclI and XbaIand inserted into a pD18 vector containing 2H7 scFv that was alsodigested with BclI and XbaI. The polynucleotide sequences for mutatedthe CH3 domains, MTCH3 Y405, MTCH3 A405, MTCH3 A407, MTCH3 Y405A407, andMTCH3 A405A407 are shown in SEQ ID NOs: 370, 371, 372, 373, 374,respectively, and the polypeptide sequences for each are shown in SEQ IDNOs: 375, 376, 377, 378, 379 respectively. The polynucleotide sequencesfor the 2H7 scFv MTH WTCH2 MTCH3 Y405, 2H7 scFv MTH WTCH2 MTCH3 A405,scFv MTH WTCH2 MTCH3 A407, scFv MTH WTCH2 MTCH3 Y405A407, and scFv MTHWTCH2 MTCH3 A405A407 are shown in SEQ ID NOs: 381, 380, 383, 384, 382,respectively, and the deduced amino acid sequences are shown in SEQ IDNOs: 386, 385, 387, 388 389, respectively.

Example 22 Construction of 2H7 SCFV IGG Fusion Proteins with HingeMutations

A 2H7 scFv IgG fusion protein was constructed with the third cysteineresidue in the IgG1 hinge region substituted with a serine residue. Thetemplate for introduction of the mutations was a polynucleotide encoding2H7 scFv WTH WTCH2CH3 (SEQ ID NO: 2, 239). The oligonucleotideintroducing the mutations was a 5′ PCR primer oligonucleotide HIgGMHcys3having the sequence 5′-gtt gtt gat cag gag ccc aaa tct tgt gac aaa actcac aca tgt cca ccg tcc cca gca cct-3′ (SEQ ID NO: 589). Theoligonucleotide introducing the mutation into the hinge region wascombined with template and a 3′ oligonucleotide containing an XbaI site(underlined and italicized) (5′-gtt gtt tct aga tca ttt acc cgg aga caggga gag get ctt ctg cgt gta g-3′ (SEQ ID NO: 278)) to amplify the mutanthinge-wild type (WT)-CH2-CH3 sequences by PCR. The IgG MTH CCS mutantsequence was amplified for 30 cycles with a denaturation profile of 94°C., annealing at 50° C. for 30 seconds, and extension at 72° C. for 30seconds. The amplified polynucleotides were inserted into the TOPO®cloning vector (Invitrogen Life Technologies) and then were sequenced asdescribed in Example 1 to confirm the presence of the mutation. pD18vector containing 2H7 scFv was digested to remove the constant regionsequences essentially as described in Example 10. The mutant hinge-wildtype CH2-CH3 regions were inserted in frame into the digested vector DNAto obtain vectors comprising 2H7 scFv MTH (CCS) WTCH2CH3 encoding DNA(SEQ ID NO: 395). The deduced polypeptide sequence is shown in SEQ IDNO: 398.

Example 23 Construction of Anti-CD20 IGE Fusion Proteins

A binding domain is fused to IgE constant region sequences such that theexpressed polypeptide is capable of inducing an allergic responsemechanism. The single chain Fv nucleotide sequence of 40.2.220 (SEQ IDNO: 466), an anti-CD40 antibody, is fused to IgE CH2-CH3-CH4 accordingto methods described for other scFv immunoglobulin constant regionconstructs (see Examples 1, 5, 10, and 13). To PCR amplify the IgECH2-CH3-CH4 domains, a 5′ oligonucleotide primer, hIgE5Bc1, having thesequence 5′-gtt gtt gat cac gtc tgc tcc agg gac ttc acc cc-3′, and a 3′oligonucleotide primer, hIgE3stop, having the sequence 5′- gtt gtt tctaga tta act ttt acc ggg att tac aga cac cgc tcg ctg g-3′ are used.

The retroviral transfection system for ectopic surface expression ofgenetically engineered cell surface receptors composed of scFvs thatbind costimulatory receptors described in Example 12 is used toconstruct a 40.2.220 scFv IgE-CD80 fusion protein. The 40.2.220 scFv IgEfusion polynucleotide sequence is fused in frame to sequences encodingthe transmembrane domain and cytoplasmic tail of human CD80 (SEQ ID NO:460), such that when the fusion protein is expressed in the transfectedcell, CD80 provided an anchor for surface expression of the scFv Igfusion protein. cDNA encoding the anti-CD40 scFv-IgE-CD80 fusionproteins is inserted into the retroviral vector pLNCX (BD BiosciencesClontech) according to standard molecular biology procedures and vendorinstructions. The 40.2.220 scFv-Ig-CD80 cDNA is inserted between the5′LTR-neomycin resistance gene-CMV promoter sequences and the 3′LTRsequence. The retroviral constructs are transfected into a carcinomacell line, and transfected cells are screened to select clones that areexpressing the 40.2.220 scFv-Ig-CD80 fusion protein on the cell surface.

Example 24 Construction of IGA-T4 Mutants that are Expressed on the CellSurface

The retroviral transfection system for ectopic surface expression ofgenetically engineered cell surface receptors composed of scFvs thatbind costimulatory receptors described in Example 12 is used toconstruct a 2H7 scFv IgA hinge IgA-T4-CD80 fusion protein. The 2H7 scFvIgAH IgA-T4 fusion polynucleotide sequence (SEQ ID NO: 298) is fused inframe to sequences encoding the transmembrane domain and cytoplasmictail of human CD80 (SEQ ID NO: 460), such that when the fusion proteinis expressed in the transfected cell, CD80 provided an anchor forsurface expression of the scFv Ig fusion protein. cDNA encoding the 2H7scFv IgAH IgA-T4-CD80 fusion protein (SEQ ID NO: 299) is inserted intothe retroviral vector pLNCX (BD Biosciences Clontech) according tostandard molecular biology procedures and vendor instructions. The 2H7scFv IgAH IgA-T4-CD80 cDNA is inserted between the 5′LTR-neomycinresistance gene-CMV promoter sequences and the 3′LTR sequence. Theretroviral construct is transfected into Reh, an acute lymphocyticleukemia cell line (ATCC CRL-8286). Transfected cells are screened toselect clones that are expressing 2H7 scFv-Ig fusion proteins on thecell surface.

Example 25 Characterization of an Anti-4-1BB scFv IG-CD80 Fusion ProteinExpressed on the Cell Surface of Tumor Cells and Growth of the TumorCells in Vivo

This example describes construction of an anti-murine 4-1BB (CD137) scFvfusion protein that has an IgG wild type hinge and CH2 and CH3 domainsthat is fused to the CD80 transmembrane and cytoplasmic domains. TheExample also illustrates the effect of the cell surface expression ofthe anti-4-1BB scFv IgG CD80 polypeptide when the transfected tumorcells are transplanted into mice.

The heavy and light chain variable regions of a rat anti-4-1BB (CD137)monoclonal antibody (1D8) were cloned, and a single chain Fv constructwas prepared essentially as described in Example 1. The heavy chain andlight chain variable regions of each antibody were cloned according tostandard methods for cloning immunoglobulin genes and as described inExample 1. Aingle chain Fv construct was prepared as described inExample 1 by inserting a nucleotide sequence encoding a (gly₄ser)₃peptide linker between the VL region nucleotide sequence of 1D8 (SEQ IDNOS: 336, 549) and the VH region nucleotide sequence of 1D8 (SEQ ID NOS:333, 547). The polypeptide sequence for 1D8 VL is shown in SEQ ID NOS:342, 550, and the polypeptide sequence for the VH domain is shown in SEQID NOS: 341, 548. The scFv polynucleotide (SEQ ID NO: 337) was thenfused to human IgG1 wild-type hinge-CH2-CH3 domains according to themethods described in Example 1. The scFv IgG1 fusion polynucleotidesequence was then fused in frame to sequences encoding the transmembranedomain and cytoplasmic tail of human CD80 (SEQ ID NO: 460) essentiallyas described in Example 12, such that when the fusion protein wasexpressed in the transfected cell, CD80 provided an anchor for surfaceexpression of the scFv Ig fusion protein. cDNA encoding thescFv-IgG-CD80 fusion protein (SEQ ID NO: 340) was inserted into theretroviral vector pLNCX (BD Biosciences Clontech) according to standardmolecular biology procedures and vendor instructions. The scFv-Ig-CD80cDNA was inserted between the 5′LTR-neomycin resistance gene-CMVpromoter sequences and the 3′LTR sequence.

The retroviral constructs were transfected into the metastatic M2 cloneof K1735, a melanoma cell line, provided by Dr. I. Hellstrom, PNRI,Seattle, Wash. Transfected cells were screened to select clones thatwere expressing scFv-Ig fusion proteins on the cell surface. Todemonstrate that the 1D8 scFv IgG-CD80 construct was expressed on thecell surface of the tumor cells, the transfected cells were analyzed byflow immunocytofluorimetry. Transfected cells (K1735-1D8) were incubatedfor one hour on ice in phycoerythrin-conjugated F(ab′)₂ goat anti-humanIgG. The unbound conjugate was then removed by washing the cells andflow cytometry analysis was performed using a Coulter Epics XL cellsorter. Results are presented in FIG. 41A.

The growth of K1735-1D8 transfected cells was examined in vivo. K1735-WTcells grew progressively when transplanted subcutaneously (s.c.) innaïve C3H mice. Although the same dose of K1735-1D8 cells initiallyformed tumors of an approximately 30 mm² surface area, the tumorsstarted to regress around day 7 and had disappeared by day 20 as shownin FIG. 41B. Tumor cells that were transfected with a similarlyconstructed vector encoding a non-binding scFv, a human anti-CD28 scFvconstruct, grew as well as tumor cells that had not been transfected.The presence of a foreign protein, that is, human IgG1 constant domainsor rat variable regions, did not make transfected K1735-WT cellsimmunogenic; the growth of the K1735-1D8 cells in C3H mice was identicalto that of K1735-WT cells (untransfected).

To investigate the roles of CD4⁺ and CD8⁺ T lymphocytes and NK cells inthe regression of K1735-1D8 tumors, na˜ve mice were injectedintraperitoneally (i.p.) with monoclonal antibodies (mAbs, typically 50μg in a volume 0.1 ml) to remove CD8⁺, CD4⁺ or both CD4⁺ and CD8⁺ Tcells, or were injected with anti-asialo-GM1 rabbit antibodies to removeNK cells. Twelve days later, when flow cytometry analysis of spleencells from identically treated mice showed that the targeted T cellpopulations were depleted, K1735-1D8 cells were transplanted s.c to eachT cell-depleted group. K1735-1D8 had similar growth kinetics in micethat had been injected with the anti-CD8 MAb or control rat IgG whileremoval of CD4⁺ T cells resulted in the growth of K1735-1D8 with thesame kinetics as K1735-WT. This failure to inhibit tumor growth afterCD4+ T cell removal was observed regardless of the presence or absenceof CD8+ T cells. K1735-1D8 grew in all NK-depleted mice, although moreslowly than in the CD4-depleted group. The results are presented in FIG.41C.

Example 26 Therapeutic Effect of Tumor Cells Expressing Anti-4-1 BB SCFVIGG-CD80 Fusion Protein

This Example examines the ability of K1735-1D8 transfected tumor cellsexpressing an anti-CD137 scFv on the cell surface to generate asufficient immune response in mice to mediate rejection of established,untransfected wild type tumors. C3H mice were transplanted with K1735-WTtumors (2×10⁶ cells/animal) and grown for approximately six days.Experiments were performed using mice with established K1735-WT tumorsof 30 mm² surface area. Mice were vaccinated by s.c. injection ofK1735-1D8 or irradiated K1735-WT cells on the contralateral side.Identical injections were repeated at the time points indicated in FIG.42. One group of animals was given four weekly injections of K1735-1D8cells. According to the same schedule, another group was givenirradiated (12,000 rads) K1735-WT cells, and a third group was injectedwith PBS. The data are plotted in FIG. 42. The WT tumors grewprogressively in all control mice and in all mice that receivedirradiated K1735-WT cells. In contrast, the tumors regressed in 4 of the5 mice treated by immunization with K1735-1D8. The animals remainedtumor-free and without signs of toxicity when the experiment wasterminated 3 months later. In the fifth mouse, the tumor noduledecreased in size as long as the mouse received K1735-1D8 cells, but thetumor grew back after therapy was terminated.

In another experiment with 5 mice/group, mice were injectedintravenously (i.v.) with 3×10⁵ K1735-WT cells to initiate lungmetastases. Three days later, K1735-1D8 cells were transplanted s.c.This procedure was repeated once weekly for a month; control mice wereinjected with PBS. The experiment was terminated when one mouse in thecontrol group died, 37 days after receiving the K1735-WT cells. At thattime, lungs of the control mice each had >500 metastatic foci. Incontrast, less than 10 metastatic foci were present in the lungs of theimmunized mice.

In a third experiment, mixtures of K1735-WT cells and K1735-1D8 cellswere injected into immunocompetent syngeneic C3H mice. Mice wereinjected subcutaneously with K7135-WT cells alone or with a mixture of2×10⁶ K1735-WT cells and 2×10⁵ K1735-1D8 cells. Tumor growth wasmonitored at 5-day intervals.

Example 27 Expression of Anti-4-1BB scFv IGG-CD80 Fusion Protein on theCell Surface of Sarcoma Cells

This Example demonstrates expression of an anti-CD 137 scFv on the cellsurface of a second type of tumor cell by transfecting a murine sarcomacell line with an anti-CD137 scFv IgG-CD80 construct.

The 1D8 scFv IgG WTH WTCH2CH3-CD80 polynucleotide (SEQ ID NO: 340) wastransferred from the pLNCX vector into pCDNA3-hygro vector usingrestriction enzyme digestion and ligation steps according to standardmolecular biology methods. The construct was cut with HindIII+Clal andthesFv fragment was filled in with Klenow (Roche) and the blunt-endedfragment was ligated into EcoR5 site of pcDNA3. Ag104 murine sarcomatumor cells were transfected with the pCDNA3-hygro vector containing the1D8 scFv IgG CD80 fusion protein. Hygromycin-resistant clones werescreened by flow cytometry using a FITC anti-human IgG antibody todetect expression of the transgene. Only approximately 15% of theresistant clones had detectable fusion protein initially. Positive cellsidentified by flow cytometry were repeatedly panned on flasks coatedwith immobilized anti-human IgG (10 μg/ml) according to standardmethods. Panning was performed by incubating cells on the coated platesfor 30 min at 37 C; the plates were then washed 2-3× in versene or PBS.After each round, cells were tested for IgG expression by FACS. Thehistogram in FIG. 44 shows the staining pattern after four rounds ofpanning against anti-human IgG (black). Untransfected cells were stainedand are indicated in gray. All of the cells in the population werepositive.

Example 28 Construction and Characterization of A Bispecific SCFV IGFusion Protein and SCFV IG Fusion Proteins with A Mutation in the IGG1CH2 Domain

An anti-CD20 (2H7) scFv IgG fusion protein was constructed that had amutant hinge (MT (SSS)) and a mutant CH2 domain in which the proline atresidue (position number 238 according to Ward et al., supra) wassubstituted with a serine. The 2H7 scFv IgG MTH (SSS) MTCH2WTCH3encoding polynucleotide (SEQ ID NO: 3, 351) was constructed essentiallyaccording to methods described in Examples 1, 5, and 13. The IgG mutanthinge-mutant CH2-wild type CH3 domains were also fused to an anti-CD20(2H7)-anti-CD40 (40.2.220) bispecific scFv. The anti-CD20-anti-CD40 scFvIgG MTH (SSS) MTCH2WTCH3 encoding polynucleotide sequence is shown inSEQ ID NO: 349 and the encoded polypeptide is shown in SEQ ID NO: 350.

COS cells were transiently transfected with vectors comprising thepolynucleotide sequences encoding 2H7 scFv IgG MTH (SSS) MTCH2WTCH3 (SEQID NO: 3, 351); anti-CD20-anti-CD40 scFv IgG MTH (SSS) MTCH2WTCH3 (SEQID NO: 349); 2H7 scFv IgG MTH (SSS) WTCH2CH3 (SEQ ID NO: 4, 225); and2H7 scFv IgAH IgG WTCH2CH3 (SEQ ID NO: 5, 283) as described in Example10. Culture supernatants were collected and the fusion proteins werepurified by protein A chromatography (see Example 10). The purifiedpolypeptides were fractionated by SDS-PAGE according to the methoddescribed in Example 10. Rituximab (anti-CD20 monoclonal antibody), andBio-Rad prestained molecular weight standards (Bio-Rad, Hercules,Calif.), and Multimark® molecular weight standards (Invitrogen LifeTechnologies were also applied to the gel. The results are presented inFIG. 45.

The 2H7 scFv Ig fusion protein that contains a mutation in the CH2domain was compared to fusion proteins that have the wild type CH2domain in an ADCC assay. The assays were performed essentially asdescribed in Examples 11 and 19. Fresh resting PBMC (effector cells)were added to ⁵¹Cr-labeled BJAB cells (target cells) at the ratiosindicated in FIG. 46. Purified 2H7 scFv IgG MTH (SSS) MTCH2WTCH3, 2H7scFv IgG MTH (SSS) WTCH2CH3, 2H7 scFv IgAH IgG WTCH2CH3, and Rituximab,each at 10 μg/ml were added to the effector/target cell mixtures andincubated for five hours at 37° C. Supernatants were harvested and theamount of chromium released was determined as described in Examples 11and 19. Percent specific killing by each fusion protein is presented inFIG. 46.

Example 29 Tumor Cell Surface Expression of an Anti-Human CD3 SCFV IGGFusion Protein

An anti-human CD3 scFv Ig CD80 fusion protein was prepared essentiallyas described in Examples 1 and 12. The fusion protein comprised ananti-human CD3 scFv fused to wild type IgG1 hinge (SEQ ID NO: 436, 438)and wild type CH2 (SEQ ID NO: 440) and CH3 (SEQ ID NO: 442) domains,fused to CD80 transmembrane and cytoplasmic domains (SEQ ID NO: 461) toenable cell surface expression of the anti-CD3 scFv. The anti-human CD3scFv IgG WTH WTCH2CH3-CD80 polynucleotide (SEQ ID NO: 559) encoding thepolypeptide (SEQ ID NO: 560) was transfected in Reh cells and into T51cells (lymphoblastoid cell line). Expression of the anti-human CD3 scFvIgG fusion protein was detected by flow cytometry using FITC conjugatedgoat anti-human IgG (see methods in Examples 4, 10, 16, 18). FIG. 47Aillustrates expression of the anti-human CD3 fusion protein on the cellsurface of Reh cells, and FIG. 47B shows expression of the fusionprotein on T41 cells.

ADCC assays were performed with the transfected Reh and T51 cells todetermine if expression of the scFv-Ig polypeptides on the cell surfaceaugmented effector cell function. Untransfected and transfected Rehcells and untransfected and transfected T51 cells were pre-labeled with⁵¹Cr (100 μCi) (Amersham) for two hours at 37° C. Human PBMC served aseffector cells and were added to the target cells (5×10⁴ cells per wellof 96 well plate) at ratios of 20:1, 10:1, 5:1, and 2.5:1. After fourhours at 37° C., culture supernatants were harvested and analyzed asdescribed in Examples 11 and 12. Percent specific killing was calculatedas described in Example 12. The results are presented in FIG. 48.

Example 30 Induction of Cytokine Expression in Tumor Cells ExpressingAnti-CD28 SCFV on the Cell Surface

This Example describes the effect of cell surface expressed scFv oncytokine mRNA induction in stimulated lymphocytes co-cultured with tumorcells transfected with an anti-human CD28 scFv IgG-CD80 fusion protein.

Real time PCR analysis was performed on RNA samples from human PBMCstimulated with Reh, Reh-anti-CD28 (2e12) (see Example 12 forconstruction of 2e12 scFv IgG WTH WHTCH3CH2-CD80 and transfection of Rehcells), and Reh-CD80 (see Example 14) in order to measure the effects ofthe surface expressed scFv on cytokine production by the PBMC effectorcells. For the real-time PCR assay, SYBR Green (QIAGEN) (Morrison etal., Biotechniques 24:954-8, 960, 962 (1998)) was used and measured byan ABI PRISM® 7000 Sequence Detection System (Applied Biosystems, FosterCity, Calif.) that measures the formation of PCR product after eachamplification cycle. Cells were harvested from cultures and total RNAprepared using QIAGEN RNA kits, including a QIA shredder columnpurification system to homogenize cell lysates, and RNeasy® mini-columnsfor purification of RNA. cDNA was reverse transcribed using equalamounts of RNA from each cell type and Superscript II ReverseTranscriptase (Life Technologies). SYBR Green real-time PCR analysis wasthen performed using the prepared cDNA as template and primer pairsspecific for cytokine gene products. The average length of the PCRproducts that were amplified ranged from 150-250 base pairs. The cDNAlevels for many activation response molecules including IFNγ, TNFα,GM-CSF, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-15, ICOSL, CD80 andCD86 were assayed. Control reference cDNAs for constitutively expressedgenes, including GAPDH, β-actin, and CD3ε were measured in each assay.The most significant induction of specific mRNA was observed for IFN-γ,and more modest induction was observed for CTLA-4 and ICOS.

Example 31 Cloning of an Anti-Human 4-1BB Antibody and Construction ofan Anti-Human 4-1BB SCFV IG Fusion Protein

A hybridoma cell line expressing a mouse anti-human monoclonal antibody(designated 5B9) was obtained from Dr. Robert Mittler, Emory UniversityVaccine Center, Atlanta, Ga. The variable heavy and light chain regionswere cloned according to known methods for cloning of immunoglobulingenes and as described herein. Cells were grown in IMDM/15% FBS(Invitrogen Life Technologies) media for several days. Cells inlogarithmic growth were harvested from cultures and total RNA preparedusing QIAGEN RNA kits, including a QIA shredder column purificationsystem to homogenize cell lysates, and RNeasy® mini-columns forpurification of RNA according to manufacturer's instructions. cDNA wasreverse transcribed using random hexamer primers and Superscript IIReverse Transcriptase (Invitrogen Life Technologies).

cDNA was anchor-tailed using terminal transferase and dGTP. PCR was thenperformed using an anchor-tail complementary primer and a primer thatannealed specifically to the antisense strand of the constant region ofeither mouse Ck (for amplification of VL) or the appropriate isotypemouse CH1 (for amplification of VH). The amplified variable regionfragments were TOPO® cloned (Invitrogen Life Technologies), and cloneswith inserts of the correct size were then sequenced. Consensus sequencefor each variable domain was determined from sequence of at least fourindependent clones. The 5B9 VL and VH polynucleotide sequences are shownin SEQ ID NOs: 355 and 354, respectively, and the deduced amino acidsequences are shown in SEQ ID NOs: 361 and 360. The scFv was constructedby a sewing PCR method using overlapping primers containing a synthetic(gly₄ser)₃ linker domain inserted between the light and heavy chainvariable regions (see Example 1). The 5B9 scFv polypeptide (SEQ ID NO:356) is encoded by the polynucleotide sequence comprising SEQ ID NO:362.

5B9 scFv polynucleotide sequence was fused in frame to thepolynucleotide sequence encoding the human IgG1 mutant hinge and wildtype CH2 and CH3 (MTH (SSS) WTCH2CH3, SEQ ID NO: 357) according tomethods described in Examples 5, 10, and 13. COS cells were transientlytransfected with a vector comprising the 5B9 scFv IgG MTH (SSS) WTCH2CH3polynucleotide sequence (SEQ ID NO: 357). Supernatant was collected andbinding of the 5B9 scFv IgG MTH (SSS) WTCH2CH3 polypeptide (SEQ ID NO:362) was measured by flow immunocytofluorimetry essentially as describedin Examples 4, 10, 16, and 18. Culture supernatant from the 5B9hybridoma cell line was also included in the binding assay. Fresh humanPBMC were incubated in the presence of immobilized anti-CD3 for fourdays prior to the binding experiment to induce expression of CD 137 onthe surface of activated T cells. Stimulated PBMC were washed andincubated with COS or hybridoma culture supernatant containing the 5B9scFv IgG fusion protein or 5B9 murine monoclonal antibody, respectively,for 1 hour on ice. Binding of 5B9 scFv IgG fusion protein or 5B9 murinemonoclonal antibody was detected with FITC conjugated anti-human IgG oranti-mouse IgG, respectively. The results are presented in FIG. 49.

Example 32 Construction of 2H7 SCFV IGG Fusion Proteins with HingeMutations

2H7 scFv IgG fusion proteins are constructed with the first cysteineresidue and the second cysteine in the IgG1 hinge region substitutedwith a serine residue to provide MTH (SCC) and MTH (CSC). The templatefor introduction of the mutations is a polynucleotide encoding 2H7 scFvWTH WTCH2CH3 (SEQ ID NO: 2, 207). The oligonucleotide introducing themutations are 5′ PCR primer oligonucleotides HIgGMHcys1 (SEQ ID NO: 587)and HIgGMHcys2 (SEQ ID NO: 391). The encoding polynucleotides of themutants are presented in SEQ ID NOs: 393, 394 and the polypeptidesequences are provided in SEQ ID NO: 396, 397.

Additional representative sequences of the present invention are asfollows:

HuIgG1 wild type hinge, CH2, CH3 (SEQ ID NO: 427)tctgatcaggagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga HuIgG1 wild typehinge, CH2, CH3 (SEQ ID NO: 428)sdqepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkLlama IgG1 hinge, CH2, CH3 (SEQ ID NO: 429)tgatcaagaaccacatggaggatgcacgtgcccncagtgcccncaatgcccngcnccngaactnccaggaggcccttctgtctttgtcttccccccgaaacccaaggacgtcctctccatttttggaggccgagtcacgtgcgttgtagtggacgtcggaaagaaagaccccgaggtcaatttcaactggtatattgatggcgttgaggtgcgaacggccaatacgaagccaaaagaggaacagttcaacagcacgtaccgcgtggtcagcgtcctgcccatccagcaccaggactggctgacggggaaggaattcaagtgcaaggtcaacaacaaagctctcccggcccccatcgagaggaccatctccaaggccaaagggcagacccgggagccgcaggtgtacaccctggccccacaccgggaagaactggccaaggacaccgtgagcgtaacatgcctggtcaaaggcttctacccagctgacatcaacgttgagtggcagaggaacggtcagccggagtcagagggcacctacgccaacacgccgccacagctggacaacgacgggacctacttcctctacagcaagctctcggtgggaaagaacacgtggcagcggggagaaaccttaacctgtgtggtgatgcatgaggccctgcacaaccactacacccagaaatccatcacccagtcttcgggtaaatagtaatctaga Llama IgG1hinge, CH2, CH3 (In FIG. 23 as Llama IgG1) (SEQ ID NO: 430)ephggctcpqcpapelpggpsvfvfppkpkdvlsisgrpevtcvvvdvgkedpevnfnwyidgvevrtantkpkeeqfnstyrvvsvlpiqhqdwltgkefkckvnnkalpapiertiskakgqtrepqvytlaphreelakdtvsvtclvkgfypadinvewqrngqpesegtyantppqldndgtyflysrlsvgkntwqrgetltgvvmhealhnhytqksitqssgkLlama IgG2: (SEQ ID NO: 431)tgatcaagaacccaagacaccaaaaccacaaccacaaccacaaccacaacccaatcctacaacagaatccaagtgtcccaaatgtccagcccctgagctcctgggagggccctcagtcttcatcttccccccgaaacccaaggacgtcctctccatttctgggaggcccgaggtcacgtgcgttgtggtagacgtgggccaggaagaccccgaggtcagtttcaactggtacattgatggcgctgaggtgcgaacggccaacacgaggccaaaagaggaacagttcaacagcacgtaccgcgtggtcagcgtcctgcccatccagcaccaggactggctgacggggaaggaattcaagtgcaaggtcaacaacaaagctctcccggcccccatcgagaagaccatctccaaggccaaagggcagacccgggagccgcaggtgtacaccctggccccacaccgggaagagctggccaaggacaccgtgagcgtaacatgcctggtcaaaggcttctacccacctgatatcaacgttgagtggcagaggaatgggcagccggagtcagagggcacytacgccaccacgccaccccagctggacaacgacgggacctacttcctctacagcaagctctcggtgggaaagaacacgtggcagcagggagaaaccttcacctgtgtggtgatgcacgaggccctgcacaaccactacacccagaaatccatcacccagtcttcgggtaaatagtaatctaga Llama IgG2 (SEQ ID NO: 432)Dqepktpkpqpqpqpqpnptteskcpkcpapellggpsvfifppkpkdvlsisgrpevtcvvvdvgqedpevsfnwyidgaevrtantrpkeeqfnstyrvvsvlpiqhqdwltgkefkckvnnkalpapiektiskakgqtrepqvytlaphreelakdtvsvtclvkgfyppdinvewqrngqpesegtyattppqldndgtyflysklsvgkntwqqgetftcvvmhealhnhytqksitqssgk Llama IgG3 Fc (SEQ ID NO: 433)tgatcaagcgcaccacagcgaagaccccagctccaagtgtcccaaatgcccaggccctgaactccttggagggcccacggtcttcatcttccccccgaaagccaaggacgtcctctccatcacccgaaaacctgaggtcacgtgcttgtggtggacgtgggtaaagaagaccctgagatcgagttcaagctggtccgtggatgacacagaggtacacacggctgagacaaagccaaaggaggaacagttcaacagcacgtaccgcgtggtcagcgtcctgcccatccagcaccaggactggctgacggggaaggaattcaagtgcaaggtcaacaacaaagctctcccagcccccatcgagaggaccatctccaaggccaaagggcagacccgggagccgcaggtgtacaccctggccccacaccgggaagagctggccaaggacaccgtgagcgtaacctgcctggtcaaaggcttcttcccagctgacatcaacgttgagtggcagaggaatgggcagccggagtcagagggcacctacgccaacacgccgccacagctggacaacgacgggacctacttcctctacagcaaactctccgtgggaaagaacacgtggcagcagggagaagtcttcacctgtgtggtgatgcacgaggctctacacaatcactccacccagaaatccatcacccagtcttcgggtaaatagtaatctagagggcccLlama IgG3 Fc (SEQ ID NO: 434)dqahhsedpsskcpkcpgpellggptvfifppkakdvlsitrkpevtclwwtwvkktlrsssswsvddtevhtaetkpkeeqfnstyrvvsvlpiqhqdwltgkefkckvnnkalpapiertiskakgqtrepqvytlaphreelakdtvsvtclvkgffpadinvewqrngqpesegtyantppqldndgtyflysklsvgkntwqqgevftcvvmhealhnhstqksitqssgkHuIgG1 wild type hinge (SEQ ID NO: 435)gatcaggagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagca HuIgG1 wild typehinge (SEQ ID NO: 436) dqepkscdkthtcppcpa HuIgG1 H2, wild type hingewith leu at second position (results from BglI site) (SEQ ID NO: 437)gatctggagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagca HuIgG1 H2, wildtype hinge with leu at second position. (SEQ ID NO: 438)dlepkscdkthtcppcpa NT HuIgG1 wild type CH2 (SEQ ID NO: 439)cctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaa HuIgG1 wild type CH2 AA (SEQ ID NO: 440)pellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskak NT HuIgG1 wild type CH3 (SEQID NO: 441)gggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatgaAA HuIgG1 wild type CH3 (SEQ ID NO: 442)gqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk NT HuIgG1 mutated hinge(C-C-C→S-S-S) (SEQ ID NO: 443)gatcaggagcccaaatcttctgacaaaactcacacatccccaccgtccccagca AA HuIgG1 mutatedhinge (C-C-C→S-S-S) (SEQ ID NO: 444) dqepkssdkthtsppspa Mutant hinge,but wild type CH2 and CH3-reads from the hinge + Ig tail, HIgG1MTHWTCH2CH3: (SEQ ID NO: 445)tgatcaccccaaatcttctgacaaaactcacacatctccaccgtcctcagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgataatctaga Protein sequence:Mutant hinge, but wild type CH2 and CH3 (SEQ ID NO: 446)dhpkssdkthtsppssapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkLLG1-5′bgl 35 mer Llama IgG1 5′ (SEQ ID NO: 447) 5′-gtt gtt gat caa gaacca cat gga gga tgc acg tg-3′ LLG2-5′bgl 32 mer, Llama IgG2-5′ (SEQ IDNO: 448) 5′-gtt gtt gat caa gaa ccc aag aca cca aaa cc-3′ LLG3-5′bgl 33mer, Llama IgG3-5′ (SEQ ID NO: 449) 5′-gtt gtt gat caa gcg cac cac agcgaa gac ccc-3′ LLseqsense 19mer, llama sequencing primer (SEQ ID NO:450) 5′-ctg aga tcg agt tca gct g-3′ LLseqAS 19 mer (SEQ ID NO: 451)5′-cct cct ttg gct ttg tct c-3′ NT 2H7 scFv llama IgG1 (SEQ ID NO: 452)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaagaaccacatggaggatgcacgtgcccncagtgcccncaatgcccngcnccngaactnccaggaggcccttctgtctttgtcttccccccgaaacccaaggacgtcctctccatttttggaggccgagtcacgtgcgttgtagtggacgtcggaaagaaagaccccgaggtcaatttcaactggtatattgatggcgttgaggtgcgaacggccaatacgaagccaaaagaggaacagttcaacagcacgtaccgcgtggtcagcgtcctgcccatccagcaccaggactggctgacggggaaggaattcaagtgcaaggtcaacaacaaagctctcccggcccccatcgagaggaccatctccaaggccaaagggcagacccgggagccgcaggtgtacaccctggccccacaccgggaagaactggccaaggacaccgtgagcgtaacatgcctggtcaaaggcttctacccagctgacatcaacgttgagtggcagaggaacggtcagccggagtcagagggcacctacgccaacacgccgccacagctggacaacgacgggacctacttcctctacagcaagctctcggtgggaaagaacacgtggcagcggggagaaaccttaacctgtgtggtgatgcatgaggccctgcacaaccactacacccagaaatccatcacccagtcttcgggtaaatagtaatctagaAA 2H7 scFv llama IgG1 (SEQ ID NO: 453)mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggssqaylqqsgaelvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvssdqephggctcpqcpapelpggpsvfvfppkpkdvlsifggrvtcvvvdvgkkdpevnfnwyidgvevrtantkpkeeqfnstyrvvsvlpiqhqdwltgkefkckvnnkalpapiertiskakgqtrepqvytlaphreelakdtvsvtclvkgfypadinvewqrngqpesegtyantppqldndgtyflysklsvgkntwqrgetltcvvmhealhnhytqksitqssgk NT 2H7 scFv llama IgG2 (SEQ ID NO: 454)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaagaacccaagacaccaaaaccacaaccacaaccacaaccacaacccaatcctacaacagaatccaagtgtcccaaatgtccagcccctgagctcctgggagggccctcagtcttcatcttccccccgaaacccaaggacgtcctctccatttctgggaggcccgaggtcacgtgcgttgtggtagacgtgggccaggaagaccccgaggtcagtttcaactggtacattgatggcgctgaggtgcgaacggccaacacgaggccaaaagaggaacagttcaacagcacgtaccgcgtggtcagcgtcctgcccatccagcaccaggactggctgacggggaaggaattcaagtgcaaggtcaacaacaaagctctcccggcccccatcgagaagaccatctccaaggccaaagggcagacccgggagccgcaggtgtacaccctggccccacaccgggaagagctggccaaggacaccgtgagcgtaacatgcctggtcaaaggcttctacccacctgatatcaacgttgagtggcagaggaatgggcagccggagtcagagggcacytacgccaccacgccaccccagctggacaacgacgggacctacttcctctacagcaagctctcggtgggaaagaacacgtggcagcagggagaaaccttcacctgtgtggtgatgcacgaggccctgcacaaccactacacccagaaatccatcacccagtcttcgggtaaatagtaatctaga AA 2H7 scFv llama IgG2 (SEQ IDNO: 455)mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggssqaylqqsgaelvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvssdqepktpkpqpqpqpqpnptteskcpkcpapellggpsvfifppkpkdvlsisgrpevtcvvvdvgqedpevsfnwyidgaevrtantrpkeeqfnstyrvvsvlpiqhqdwltgkefkckvnnkalpapiektiskakgqtrepqvytlaphreelakdtvsvtclvkgfyppdinvewqrngqpesegtyattppqldndgtyflysklsvgkntwqqgetftcvvmhealhnhytqksitqssgk NT 2H7 scFv llama IgG3 (SEQ ID NO: 456)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaagcgaaccacagcgaagaccccagctccaagtgtcccaaatgcccaggccctgaactccttggagggcccacggtcttcatcttccccccgaaagccaaggacgtcctctccatcacccgaaaacctgaggtcacgtgcttgtggtggacgtgggtaaagaagaccctgagatcgagttcaagctggtccgtggatgacacagaggtacacacggctgagacaaagccaaaggaggaacagttcaacagcacgtaccgcgtggtcagcgtcctgcccatccagcaccaggactggctgacggggaaggaattcaagtgcaaggtcaacaacaaagctctcccagcccccatcgagaggaccatctccaaggccaaagggcagacccgggagccgcaggtgtacaccctggccccacaccgggaagagctggccaaggacaccgtgagcgtaacctgcctggtcaaaggcttcttcccagctgacatcaacgttgagtggcagaggaatgggcagccggagtcagagggcacctacgccaacacgccgccacagctggacaacgacgggacctacttcctctacagcaaactctccgtgggaaagaacacgtggcagcagggagaagtcttcacctgtgtggtgatgcacgaggctctacacaatcactccacccagaaatccatcacccagtcttcgggtaaatagtaatctagagggccc AA 2H7 scFv llama IgG3 (SEQ ID NO: 457)mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggssqaylqqsgaelvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvssdqahshsedpsskcpkcpgpellggptvfifppkakdvlsitrkpevtclwwtwvkktlrsssswsvddtevhtaetkpkeeqfnstyrvvsvlpiqhqdwltgkefkckvnnkalpapiertiskakgqtrepqvytlaphreelakdtvsvtclvkgffpadinvewqrngqpesegtyantppqldndgtyflysklsvgkntwqqgevftcvvmhealhnhstqksitqssgk 2H7 + Completely WT IgG tail: 2H7 scFv WTH WTCH2CH3(SEQ ID NO: 458) Nucleotide sequence:aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga2H7 + Completely WT IgG tail: 2H7 scFv WTH WTCH2CH3 (SEQ ID NO: 459)Protein sequencemdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggssqaylqqsgaelvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvssdqepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk NT CD80 transmembrane domain and cytoplasmic tail(+restriction sites) (SEQ ID NO: 460)gcggatccttcgaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgatAA CD80 transmembrane domain and cytoplasmic tail (SEQ ID NO: 461)adpsnllpswaitlisvngifviccltycfaprcrerrrnerlrresvrpv NT 40.2.220 VL(anti-human CD40 scFv #1--VL) (SEQ ID NO: 462)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgttctgactcagtctccagccaccctgtctgtgactccaggagatagagtctctctttcctgcagggccagccagagtattagcgactacttacactggtatcaacaaaaatcacatgagtctccaaggcttctcatcaaatatgcttcccattccatctctgggatcccctccaggttcagtggcagtggatcagggtcagatttcactctcagtatcaacagtgtggaacctgaagatgttggaatttattactgtcaacatggtcacagctttccgtggacgttcggtggaggcaccaagctggaaatcaaacgg AA 40.2.220 VL(anti-human CD40 scFv #1--VL) (SEQ ID NO: 463)mdfqvqifsfllisasvimsrgvdivltqspatlsvtpgdrvslscrasqsisdylhwyqqkshesprllikyashsisgipsrfsgsgsgsdftlsinsvepedvgiyycqhghsfpwtfgggtkleikr NT 40.2.220VH (for anti-human CD40 scFv #1--VH) (SEQ ID NO: 464)cagatccagttggtgcaatctggacctgagctgaagaagcctggagagacagtcaggatctcctgcaaggcttctgggtatgccttcacaactactggaatgcagtgggtgcaagagatgccaggaaagggtttgaagtggattggctggataaacaccccactctggagtgccaaaatatgtagaagacttcaaggacggtttgccttctctttggaaacctctgccaacactgcatatttacagataagcaacctcaaagatgaggacacggctacgtatttctgtgtgagatccgggaatggtaactatgacctggcctactttgcttactggggccaagggacactggtcactgtctctgatca AA 40.2.220 VH (for anti-human CD40scFv #1--VH) (SEQ ID NO: 465)qiqlvqsgpelkkpgetvrisckasgyaftttgmqwvqempgkglkwigwintplwsakicrrlqgrfafsletsantaylqisnlkdedtatyfcvrsgngnydlayfaywgqgtlvtvs NT 40.2.220 scFv(anti-human CD40 scFv #1) (SEQ ID NO: 466)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgttctgactcagtctccagccaccctgtctgtgactccaggagatagagtctctctttcctgcagggccagccagagtattagcgactacttacactggtatcaacaaaaatcacatgagtctccaaggcttctcatcaaatatgcttcccattccatctctgggatcccctccaggttcagtggcagtggatcagggtcagatttcactctcagtatcaacagtgtggaacctgaagatgttggaatttattactgtcaacatggtcacagctttccgtggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcagatccagttggtgcaatctggacctgagctgaagaagcctggagagacagtcaggatctcctgcaaggcttctgggtatgccttcacaactactggaatgcagtgggtgcaagagatgccaggaaagggtttgaagtggattggctggataaacaccccactctggagtgccaaaatatgtagaagacttcaaggacggtttgccttctctttggaaacctctgccaacactgcatatttacagataagcaacctcaaagatgaggacacggctacgtatttctgtgtgagatccgggaatggtaactatgacctggcctactttgcttactggggccaagggacactggtcactgtctctgatca AA 40.2.220 scFv (anti-human CD40scFv #1) (SEQ ID NO: 467)mdfqvqifsfllisasvimsrgvdivltqspatlsvtpgdrvslscrasqsisdylhwyqqkshesprllikyashsisgipsrfsgsgsgsdftlsinsvepedvgiyycqhghsfpwtfgggtkleikrggggsggggsggggsqiqlvqsgpelkkpgetvrisckasgyaftttgmqwvqempgkglkwigwintplwsakicrrlqgrfafsletsantaylqisnlkdedtatyfcvrsgngnydlayfaywgqgtlvtvs NT 2e12 VL (with L6 VK leader peptide) (SEQ IDNO: 468)atggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacgg AA2e12 VL (with L6 VK leader peptide) (SEQ ID NO: 469)mdfqvqifsfllisasvimsrgvdivltqspaslavslgqratiscrasesveyyvtslmqwyqqkpgqppkllisaasnvesgvparfsgsgsgtdfslnihpveeddiamyfcqqsrkvpwtfgggtkleikr NT2e12 VH (no leader peptide) (SEQ ID NO: 470)caggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgccagagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctca(gatctg) AA 2e12 VH (SEQ ID NO: 471)qvqlkesgpglvapsqslsitctvsgfsltgygvnwvrqppgkglewlgmiwgdgstdynsalksrlsitkdnsksqvflkmnslqtddtaryycardgysnfhyyvmdywgqgtsvtvss NT2e12scFv(+Restriction sites) (SEQ ID NO: 472)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgccagagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctct(gatcag) AA 2e12scFv(SEQ ID NO: 473)mdfqvqifsfllisasvimsrgvdivltqspaslavslgqratiscrasesveyyvtslmqwyqqkpgqppkllisaasnvesgvparfsgsgsgtdfslnihpveeddiamyfcqqsrkvpwtfgggtkleikrggggsggggsggggsqvqlkesgpglvapsqslsitctvsgfsltgygvnwvrqppgkglewlgmiwgdgstdynsalksrlsitkdnsksqvflkmnslqtddtaryycardgysnfhyyvmdywgqgtsvtvss 10A8 is anti-CD152 (CTLA-4) 10A8 VL(with L6 VK leader peptide) (SEQ ID NO: 474)atggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacatccagatgacacagtctccatcctcactgtctgcatctctgggaggcaaagtcaccatcacttgcaaggcaagccaagacattaagaagtatataggttggtaccaacacaagcctggaaaaggtcccaggctgctcatatattacacatctacattacagccaggcatcccatcaaggttcagtggaagtgggtctgggagagattattccctcagcatcagaaacctggagcctgaagatattgcaacttattattgtcaacagtatgataatcttccattgacgttcggctcggggacaaagttggaaataaaacgg AA 10A8 VL (SEQID NO: 475)mdfqvqifsfllisasvimsrgvdiqmtqspsslsaslggkvtitckasqdikkyigwyqhkpgkgprlliyytstlqpgipsrfsgsgsgrdyslsirnlepediatyycqqydnlpltfgsgtkleikr NT 10A8 VH(no leader peptide) (SEQ ID NO: 476)gatgtacagcttcaggagtcaggacctggcctcgtgaaaccttctcagtctctgtctctcacctgctctgtcactggctactccatcaccagtggtttctactggaactggatccgacagtttccgggaaacaaactggaatggatgggccacataagccacgacggtaggaataactacaacccatctctcataaatcgaatctccatcactcgtgacacatctaagaaccagtttttcctgaagttgagttctgtgactactgaggacacagctacatatttctgtgcaagacactacggtagtagcggagctatggactactggggtcaaggaacctcagtcaccgtctcctctgatca AA 10A8 VH (SEQ ID NO: 477)dvqlqesgpglvkpsqslsltcsvtgysitsgfywnwirqfpgnklewmghishdgrnnynpslinrisitrdtsknqfflklssvttedtatyfcarhygssgamdywgqgtsvtvss NT 10A8 scFv (SEQ IDNO: 478)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacatccagatgacacagtctccatcctcactgtctgcatctctgggaggcaaagtcaccatcacttgcaaggcaagccaagacattaagaagtatataggttggtaccaacacaagcctggaaaaggtcccaggctgctcatatattacacatctacattacagccaggcatcccatcaaggttcagtggaagtgggtctgggagagattattccctcagcatcagaaacctggagcctgaagatattgcaacttattattgtcaacagtatgataatcttccattgacgttcggctcggggacaaagttggaaataaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgatgtacagcttcaggagtcaggacctggcctcgtgaaaccttctcagtctctgtctctcacctgctctgtcactggctactccatcaccagtggtttctactggaactggatccgacagtttccgggaaacaaactggaatggatgggccacataagccacgacggtaggaataactacaacccatctctcataaatcgaatctccatcactcgtgacacatctaagaaccagtttttcctgaagttgagttctgtgactactgaggacacagctacatatttctgtgcaagacactacggtagtagcggagctatggactactggggtcaaggaacctcagtcaccgtctcctctgatca AA 10A8 scFv (SEQ ID NO: 479)mdfqvqifsfllisasvimsrgvdiqmtqspsslsaslggkvtitckasqdikkyigwyqhkpgkgprlliyytstlqpgipsrfsgsgsgrdyslsirnlepediatyycqqydnlpltfgsgtkleikrggggsggggsggggsdvqlqesgpglvkpsqslsltcsvtgysitsgfywnwirqfpgnklewmghishdgrnnynpslinrisitrdtsknqfflklssvttedtatyfcarhygssgamdywgqgtsvtvssd NT 40.2.220-hmtIgG1-hCD80 (SEQ ID NO: 480)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgttctgactcagtctccagccaccctgtctgtgactccaggagatagagtctctctttcctgcagggccagccagagtattagcgactacttacactggtatcaacaaaaatcacatgagtctccaaggcttctcatcaaatatgcttcccattccatctctgggatcccctccaggttcagtggcagtggatcagggtcagatttcactctcagtatcaacagtgtggaacctgaagatgttggaatttattactgtcaacatggtcacagctttccgtggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcagatccagttggtgcaatctggacctgagctgaagaagcctggagagacagtcaggatctcctgcaaggcttctgggtatgccttcacaactactggaatgcagtgggtgcaagagatgccaggaaagggtttgaagtggattggctggataaacaccccactctggagtgccaaaatatgtagaagacttcaaggacggtttgccttctctttggaaacctctgccaacactgcatatttacagataagcaacctcaaagatgaggacacggctacgtatttctgtgtgagatccgggaatggtaactatgacctggcctactttgcttactggggccaagggacactggtcactgtctctgatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgat AA 40.2.220-hmtIgG1-hCD80(SEQ ID NO: 481)mdfqvqifsfllisasvimsrgvdivltqspatlsvtpgdrvslscrasqsisdylhwyqqkshesprllikyashsisgipsrfsgsgsgsdftlsinsvepedvgiyycqhghsfpwtfgggtkleikrggggsggggsggggsqiqlvqsgpelkkpgetvrisckasgyaftttgmqwvqempgkglkwigwintplwsakicrrlqgrfafsletsantaylqisnlkdedtatyfcvrsgngnydlayfaywgqgtlvtvsdlepkssdkthtsppspapellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkadpsnllpswaitlisvngifviccltycfaprcrerrrnerlrresvrpv NT2e12scFv-hmtIgG1-CD80 fusion protein (SEQ ID NO: 482)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgccagagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctcagatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgat AA2e12scFv-hmtIgG1-CD80 fusion protein (SEQ ID NO: 483)mdfqvqifsfllisasvimsrgvdivltqspaslavslgqratiscrasesveyyvtslmqwyqqkpgqppkllisaasnvesgvparfsgsgsgtdfslnihpveeddiamyfcqqsrkvpwtfgggtkleikrggggsggggsggggsqvqlkesgpglvapsqslsitctvsgfsltgygvnwvrqppgkglewlgmiwgdgstdynsalksrlsitkdnsksqvflkmnslqtddtaryycardgysnfhyyvmdywgqgtsvtvssdlepkssdkthtsppspapellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkadpsnllpswaitlisvngifviccltycfaprcrerrrnerlrresvrpvNT 10A8 scFv-hmtIgG1-CD80 (SEQ ID NO: 484)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacatccagatgacacagtctccatcctcactgtctgcatctctgggaggcaaagtcaccatcacttgcaaggcaagccaagacattaagaagtatataggttggtaccaacacaagcctggaaaaggtcccaggctgctcatatattacacatctacattacagccaggcatcccatcaaggttcagtggaagtgggtctgggagagattattccctcagcatcagaaacctggagcctgaagatattgcaacttattattgtcaacagtatgataatcttccattgacgttcggctcggggacaaagttggaaataaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgatgtacagcttcaggagtcaggacctggcctcgtgaaaccttctcagtctctgtctctcacctgctctgtcactggctactccatcaccagtggtttctactggaactggatccgacagtttccgggaaacaaactggaatggatgggccacataagccacgacggtaggaataactacaacccatctctcataaatcgaatctccatcactcgtgacacatctaagaaccagtttttcctgaagttgagttctgtgactactgaggacacagctacatatttctgtgcaagacactacggtagtagcggagctatggactactggggtcaaggaacctcagtcaccgtctcctctgatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgat AA 10A8 scFv-hmtIgG1-CD80 (SEQ IDNO: 485)mdfqvqifsfllisasvimsrgvdiqmtqspsslsaslggkvtitckasqdikkyigwyqhkpgkgprlliyytstlqpgipsrfsgsgsgrdyslsirnlepediatyycqqydnlpltfgsgtkleikrggggsggggsggggsdvqlqesgpglvkpsqslsltcsvtgysitsgfywnwirqfpgnklewmghishdgrnnynpslinrisitrdtsknqfflklssvttedtatyfcarhygssgamdywgqgtsvtvssdlepkssdkthtsppspapellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkadpsnllpswaitlisvngifviccltycfaprcrerrrnerlrresvrpv NT500A2-hmtIgG1-CD80 (SEQ ID NO: 486)atgttgtatacatctcagctccttgggcttttactcttctggatttcagcctccagaagtgacatagtgctgactcagactccagccactctgtctctaattcctggagaaagagtcacaatgacctgtaagaccagtcagaatattggcacaatcttacactggtatcaccaaaaaccaaaggaggctccaagggctctcatcaagtatgcttcgcagtccattcctgggatcccctccagattcagtggcagtggttcggaaacagatttcactctcagcatcaataacctggagcctgatgatatcggaatttattactgtcaacaaagtagaagctggcctgtcacgttcggtcctggcaccaagctggagataaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcaggtcaagctgcagcagtccggttctgaactagggaaacctggggcctcagtgaaactgtcctgcaagacttcaggctacatattcacagatcactatatttcttgggtgaaacagaagcctggagaaagcctgcagtggataggaaatgtttatggtggaaatggtggtacaagctacaatcaaaaattccagggcaaggccacactgactgtagataaaatctctagcacagcctacatggaactcagcagcctgacatctgaggattctgccatctattactgtgcaagaaggccggtagcgacgggccatgctatggactactggggtcaggggatccaagttaccgtctcctctgatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgat AA 500A2-hmtIgG1-CD80 (SEQ ID NO: 487)mlytsqllglllfwisasrsdivltqtpatlslipgervtmtcktsqnigtilhwyhqkpkeapralikyasqsipgipsrfsgsgsetdftlsinnlepddigiyycqqsrswpvtfgpgtkleikrggggsggggsggggsqvklqqsgselgkpgasvklscktsgyiftdhyiswvkqkpgeslqwignvyggnggtsynqkfqgkatltvdkisstaymelssltsedsaiyycarrpvatghamdywgqgiqvtvssdlepkssdkthtsppspapellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkadpsnllpswaitlisvngifviccltycfaprcrerrrnerlrresvrpv NT 2H7 scFvMTH(SSS)WTCH2CH3 (SEQ ID NO: 488)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga2H7 scFv MTH(SSS)WTCH2CH3 protein sequence: (SEQ ID NO: 489)mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggssqaylqqsgaelvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvssdqepkssdkthtsppspapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk HuIgGMHncs1 (oligo for CSS) (SEQ ID NO: 490) gtt gttgat cag gag ccc aaa tct tgt gac aaa act cac HuIgGMHncs2 (oligo for SCS= ncs2) (SEQ ID NO: 491) gtt gtt gat cag gag ccc aaa tct tct gac aaa actcac aca tgc cca ccg HuIgGMHncs3 (oligo for SSC = ncs3) (SEQ ID NO: 492)gtt gtt gat cag gag ccc aaa tct tct gac aaa act cac aca tct cca ccg tgccca gca cct g hIgGWT3xba (3′ oligo for above mutation introduction) (SEQID NO: 493) gtt gtt tct aga tca ttt acc cgg aga cag gga gag gct ctt ctgcgt gta g Vhser11: (oligo for Leu to Ser at VH11) (SEQ ID NO: 494) ggaggt ggg agc tct cag gct tat cta cag cag tct ggg gct gag tcg gtg agg cchuIgG1-3′ (3′ oligo to amplify IgG1 C regions, 3′ end of CH3) (SEQ IDNO: 495) gtc tct aga cta tca ttt acc cgg aga cag huIgA/Gchim5 (oligo forpcr#1) (SEQ ID NO: 496) cca tct ccc tca act cca cct acc cca tct ccc tcatgc gca cct gaa ctc ctg huIgAhg-5′ (oligo for pcr#2) (SEQ ID NO: 497)gtt gtt gat cag cca gtt ccc tca act cca cct acc cca tct ccc caa cthuIgA3′ (SEQ ID NO: 498) gtt gtt tct aga tta tca gta gca ggt gcc gtc cacctc cgc cat gac aac 2H7 scFv IgAH IGG WT CH2CH3, 2H7 scFv with IgA hingeand WT CH2 and CH3 (SEQ ID NO: 499)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctctgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgcgcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga2H7 scFv IgAH IGG WT CH2CH3 protein sequence (SEQ ID NO: 500)mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggssqaylqqsgaelvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvsdqpvpstpptpspstpptpspscapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk NT 2H7 scFv IgAH IgACH2CH3 (2H7 scFv IgAhinge andIgA CH2 and CH3) (SEQ ID NO: 501)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtcatggcggaggtggacggcacctgctactgataatctaga AA 2H7 scFv IgAHIgACH2CH3 (2H7 scFv IgA hinge and IgA CH2 and CH3) (SEQ ID NO: 502)mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggssqaylqqsgaelvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvssdqpvpstpptpspstpptpspscchprlslhrpaledlllgseailtctltglrdasgvtftwtpssgksavqgppdrdlcgcysvssvlpgcaepwnhgktftctaaypesktpltatlsksgntfrpevhllpppseelalnelvtltclargfspkdvlvrwlqgsqelprekyltwasrqepsqgtttfavtsilrvaaedwkkgdtfscmvghealplaftqktidrlagkpthvnvsvvmaevdgtcy IgA hinge-CH2—CH3 (Human IgA tail, full length)(SEQ ID NO: 503)tgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtcatggcggaggtggacggcacctgctactgataatctaga IgA hinge-CH2—CH3 Protein sequence, (HumanIgA tail, full length) (SEQ ID NO: 504)Dqpvpstpptpspstpptpspscchprlslhrpaledlllgseailtctltglrdasgvtftwtpssgksavqgppdrdlcgcysvssvlpgcaepwnhgktftctaaypesktpltatlsksgntfrpevhllpppseelalnelvtltclargfspkdvlvrwlqgsqelprekyltwasrqepsqgtttfavtsilrvaaedwkkgdtfscmvghealplaftqktidrlagkpthvnvsvvmaevdgtcy Human J Chain: (SEQ ID NO: 505)agatctcaagaagatgaaaggattgttcttgttgacaacaaatgtaagtgtgcccggattacttccaggatcatccgttcttccgaagatcctaatgaggacattgtggagagaaacatccgaattattgttcctctgaacaacagggagaatatctctgatcccacctcaccattgagaaccagatttgtgtaccatttgtctgacctcagctgtaaaaaatgtgatcctacagaagtggagctggataatcagatagttactgctacccagagcaatatctgtgatgaagacagtgctacagagacctgctacacttatgacagaaacaagtgctacacagctgtggtcccactcgtatatggtggtgagaccaaaatggtggaaacagccttaaccccagatgcctgctatcctgactaatctaga Human J Chain polypeptide (SEQ ID NO: 506)rsqederivlvdnkckcaritsriirssedpnediverniriivplnnrenisdptsplrtrfvyhlsdlsckkcdpteveldnqivtatqsnicdedsatetcytydrnkcytavvplvyggetkmvetaltpdacypHUJCH5nl (J chain 5′ primer) (SEQ ID NO: 507) gtt gtt aga tct caa gaagat gaa agg att gtt ctt HUJCH3 (J chain 3′ primer-antisense) (SEQ ID NO:508) gtt gtt tct aga tta gtc agg ata gca ggc atc tgg 4 carboxy terminalamino acids deleted from IgA CH3 (SEQ ID NO: 509) GTCY IgAH IgAT4 HumanIgA tail, truncated (3T1)-(missing last 4 amino acids from carboxyterminus) (SEQ ID NO: 510)tgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtcatggcggaggtggactgataatctaga IgAH IgAT4 Protein sequence: (SEQ ID NO: 511)Dqpvpstpptpspstpptpspscchprlslhrpaledlllgseailtctltglrdasgvtftwtpssgksavqgppdrdlcgcysvssvlpgcaepwnhgktftctaaypesktpltatlsksgntfrpevhllpppseelalnelvtltclargfspkdvlvrwlqgsqelprekyltwasrqepsqgtttfavtsilrvaaedwkkgdtfscmvghealplaftqktidrlagkpthvnvsvvmaevd HUIGA3T1 (Oligo 3′: to delete 4 amino acids at carboxy end of IgACH3) (SEQ ID NO: 512) gtt gtt tct aga tta tca gtc cac ctc cgc cat gacaac aga cac HUIGA3T2: (oligo to delete 14 aa at end of IgA-T4) (SEQ IDNO: 513) gtt gtt tct aga tta tca ttt acc cgc caa gcg gtc gat ggt ctt NT2H7 scFv IgAH IgAT4 (SEQ ID NO: 514) (2H7 scFv IgA 3T1construct)--truncates the CH3 domain at the 3′endaagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtcatggcggaggtggactgataatctaga AA 2H7 scFv IgAH-T4 (SEQ ID NO:515)mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggssqaylqqsgaelvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvssdqpvpstpptpspstpptpspscchprlslhrpaledlllgseailtctltglrdasgvtftwtpssgksavqgppdrdlcgcysvssvlpgcaepwnhgktftctaaypesktpltatlsksgntfrpevhllpppseelalnelvtltclargfspkdvlvrwlqgsqelprekyltwasrqepsqgtttfavtsilrvaaedwkkgdtfscmvghealplaftqktidrlagkpthvnvsvvmaevd 14 amino acids deleted from IgAH-T4 (so that totalof 18 amino acids deleted from wild type IgA CH3 (SEQ ID NO: 516)PTHVNVSVVMAEVD IgAH IgA-T18 (Human IgA Tail truncated, 3T2) (SEQ ID NO:517)Tgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaa IgAH IgA-T18 Proteinsequence: (SEQ ID NO: 518)dqpvpstpptpspstpptpspscchprlslhrpaledlllgseailtctltglrdasgvtftwtpssgksavqgppdrdlcgcysvssvlpgcaepwnhgktftctaaypesktpltatlsksgntfrpevhllpppseelalnelvtltclargfspkdvlvrwlqgsqelprekyltwasrqepsqgtttfavtsilrvaaedwkkgdtfscmvghealplaftqktidrlagkNT 2H7 scFv IgAH IgAT18: (Human IgA Tail truncated, 3T2.) (SEQ ID NO:519)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaa AA:2H7 scFv IgAH IgAT18: (SEQ ID NO: 520)mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggssqaylqqsgaelvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvssdqpvpstpptpspstpptpspscchprlslhrpaledlllgseailtctltglrdasgvtftwtpssgksavqgppdrdlcgcysvssvlpgcaepwnhgktftctaaypesktpltatlsksgntfrpevhllpppseelalnelvtltclargfspkdvlvrwlqgsqelprekyhwasrqepsqgtttfavtsilrvaaedwkkgdtfscmvghealplaftqktidrlagk CTLA-4 IgG WTH WTCH2CH3 (Human-oncoMLP-CTLA4EC-hIgGWT) (SEQ IDNO: 521) Nucleotide sequence:gcaacctacatgatggggaatgagttgaccttcctagatgattccatctgcacgggcacctccagtggaaatcaagtgaacctcactatccaaggactgagggccatggacacgggactctacatctgcaaggtggagctcatgtacccaccgccatactacctgggcataggcaacggaacccagatttatgtaattgatccagaaccgtgcccagattctgatcaacccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgaCTLA-4 IgG WTH WTCH2CH3 Protein sequence: (SEQ ID NO: 522)mgvlltqrtllslvlallfpsmasmamhvaqpavvlassrgiasfvceyaspgkatevrvtvlrqadsqvtevcaatymmgneltflddsictgtssgnqvnltiqglramdtglyickvelmypppyylgigngtqiyvidpepcpdsdqpkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk HumanOncoM leader Peptide + CTLA4 EC (BclI) (SEQ ID NO: 523)Atgggggtactgctcacacagaggacgctgctcagtctggtccttgcactcctgtttccaagcatggcgagcatggcaatgcacgtggcccagcctgctgtggtactggccagcagccgaggcatcgccagctttgtgtgtgagtatgcatctccaggcaaagccactgaggtccgggtgacagtgcttcggcaggctgacagccaggtgactgaagtctgtgcggcaacctacatgatggggaatgagttgaccttcctagatgattccatctgcacgggcacctccagtggaaatcaagtgaacctcactatccaaggactgagggccatggacacgggactctacatctgcaaggtggagctcatgtacccaccgccatactacctgggcataggcaacggaacccagatttatgtaattgatccagaaccgtgcccagattctgatcaa Human OncoM leader Peptide+ CTLA4 EC Peptide sequence: (SEQ ID NO: 524)mgvlltqrtllslvlallfpsmasmamhvaqpavvlassrgiasfvceyaspgkatevrvtvlrqadsqvtevcaatymmgneltflddsictgtssgnqvnltiqglramdtglyickvelmypppyylgigngtqiyvidpepcpdsdqHuman OncoM leader peptide nucleotide (SEQ ID NO: 525)atgggggtactgctcacacagaggacgctgctcagtctggtccttgcactcctgtttccaagcatggcgagcatg Human OncoM leader peptide sequence: (SEQ ID NO: 526)Mgvlltqrtllslvlallfpsm NT Human CTLA4 EC (no LP) (SEQ ID NO: 527)Gcaatgcacgtggcccagcctgctgtggtactggccagcagccgaggcatcgccagctttgtgtgtgagtatgcatctccaggcaaagccactgaggtccgggtgacagtgcttcggcaggctgacagccaggtgactgaagtctgtgcggcaacctacatgacggggaatgagttgaccttcctagatgattccatctgcacgggcacctccagtggaaatcaagtgaacctcactatccaaggactgagggccatggacacgggactctacatctgcaaggtggagctcatgtacccaccgccatactacctgggcataggcaacggaacccagatttatgtaattgatccagaaccgtgcccagattct AA Human CTLA4 EC (no LP)(SEQ ID NO: 528)Amhvaqpavvlassrgiasfvceyaspgkatevrvtvlrqadsqvtevcaatymtgneltflddsictgtssgnqvnltiqglramdtglyickvelmypppyylgigngtqiyvidpepcpds NT Human CTLA4 IgGMTH (SSS) MTCH2CH3 (SEQ ID NO: 529)Atgggggtactgctcacacagaggacgctgctcagtctggtccttgcactcctgtttccaagcatggcgagcatggcaatgcacgtggcccagcctgctgtggtactggccagcagccgaggcatcgccagctttgtgtgtgagtatgcatctccaggcaaagccactgaggtccgggtgacagtgcttcggcaggctgacagccaggtgactgaagtctgtgcggcaacctacatgatggggaatgagttgaccttcctagatgattccatctgcacgggcacctccagtggaaatcaagtgaacctcactatccaaggactgagggccatggacacgggactctacatctgcaaggtggagctcatgtacccaccgccatactacctgggcataggcaacggaacccagatttatgtaattgatccagaaccgtgcccagattctgatcaacccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga AA Human CTLA4IgG MTH (SSS) MTCH2CH3 (SEQ ID NO: 530)Mgvlltqrtllslvlallfpsmasmamhvaqpavvlassrgiasfvceyaspgkatevrvtvlrqadsqvtevcaatymmgneltflddsictgtssgnqvnltiqglramdtglyickvelmypppyylgigngtqiyvidpepcpdsdqpkssdkthtsppspapellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk CTLA-4IgAH IgACH2CH3 (Human-oncoMLP-CTLA4EC-IgA) (SEQ ID NO: 531) Nucleotidesequence:atgggggtactgctcacacagaggacgctgctcagtctggtccttgcactcctgtttccaagcatggcgagcatggcaatgcacgtggcccagcctgctgtggtactggccagcagccgaggcatcgccagctttgtgtgtgagtatgcatctccaggcaaagccactgaggtccgggtgacagtgcttcggcaggctgacagccaggtgactgaagtctgtgcggcaacctacatgatggggaatgagttgaccttcctagatgattccatctgcacgggcacctccagtggaaatcaagtgaacctcactatccaaggactgagggccatggacacgggactctacatctgcaaggtggagctcatgtacccaccgccatactacctgggcataggcaacggaacccagatttatgtaattgatccagaaccgtgcccagattctgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtcatggcggaggtggacggcacctgctactgataatctaga CTLA-4 IgAH IgACH2CH3 Proteinsequence: (SEQ ID NO: 532)mgvlltqrtllslvlallfpsmasmamhvaqpavvlassrgiasfvceyaspgkatevrvtvlrqadsqvtevcaatymmgneltflddsictgtssgnqvnltiqglramdtglyickvelmypppyylgigngtqiyvidpepcpdsdqpvpstpptpspstpptpspscchprlslhrpaledlllgseailtctltglrdasgvtftwtpssgksavqgppdrdlcgcysvssvlpgcaepwnhgktftctaaypesktpltatlsksgntfrpevhllpppseelalnelvtltclargfspkdvlvrwlqgsqelprekyltwasrqepsqgtttfavtsilrvaaedwkkgdtfscmvghealplaftqktidrlagkpthvnvsvvmaevdgtcyCTLA-4 IgAH IgA-T4 (Human-oncoMLP-CTLA4EC-IgA3T1) (SEQ ID NO: 533)Nucleotide sequence:atgggggtactgctcacacagaggacgctgctcagtctggtccttgcactcctgtttccaagcatggcgagcatggcaatgcacgtggcccagcctgctgtggtactggccagcagccgaggcatcgccagctttgtgtgtgagtatgcatctccaggcaaagccactgaggtccgggtgacagtgcttcggcaggctgacagccaggtgactgaagtctgtgcggcaacctacatgatggggaatgagttgaccttcctagatgattccatctgcacgggcacctccagtggaaatcaagtgaacctcactatccaaggactgagggccatggacacgggactctacatctgcaaggtggagctcatgtacccaccgccatactacctgggcataggcaacggaacccagatttatgtaattgatccagaaccgtgcccagattctgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtcatggcggaggtggactgataatctaga CTLA-4 IgAH IgA-T4 Protein sequence: (SEQ IDNO: 534)MgvlltqrtllslvlallfpsmasmamhvaqpavvlassrgiasfvceyaspgkatevrvtvlrqadsqvtevcaatymmgneltflddsictgtssgnqvnltiqglramdtglyickvelmypppyylgigngtqiyvidpepcpdsdqpvpstpptpspstpptpspscchprlslhrpaledlllgseailtctltglrdasgvtftwtpssgksavqgppdrdlcgcysvssvlpgcaepwnhgktftctaaypesktpltatlsksgntfrpevhllpppseelalnelvtltclargfspkdvlvrwlqgsqelprekyltwasrqepsqgtttfavtsilrvaaedwkkgdtfscmvghealplaftqktidrlagkpthvnvsvvmaevdNT human IgG1 CH2 with 238 mutation pro→ser (SEQ ID NO: 535)cctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaag AA human IgG1 CH2 with 238 mutation pro→ser (SEQ ID NO: 536)pellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskak Amino acids surrounding Pro toSer in CH2 (SEQ ID NO: 537) PAPELLGGPS Amino acids surrounding Pro toSer in CH2 (SEQ ID NO: 538) PAPELLGGSS HIgE5Bcl (SEQ ID NO: 539) gtt gttgat cac gtc tgc tcc agg gac ttc acc cc hIgE3stop (SEQ ID NO: 540) gttgtt tct aga tta act ttt acc ggg att tac aga cac cgc tcg ctg g hIgE3BB(leaves an open reading frame at end of gene to read into transmembraneand cytoplasmic tail domain attached at either the BamHI or SfuI sites)(SEQ ID NO: 541) gtt gtt ttc gaa gga tcc gct tta ccg gga ttt aca gac accgct cgc tgg NT human IgE Fc (CH2—CH3—CH4) ORF: (SEQ ID NO: 542)tgatcacgtctgctccagggacttcaccccgcccaccgtgaagatcttacagtcgtcctgcgacggcggcgggcacttccccccgaccatccagctcctgtgcctcgtctctgggtacaccccagggactatcaacatcacctggctggaggacgggcaggtcatggacgtggacttgtccaccgcctctaccacgcaggagggtgagctggcctccacacaaagcgagctcaccctcagccagaagcactggctgtcagaccgcacctacacctgccaggtcacctatcaaggtcacacctttgaggacagcaccaagaagtgtgcagattccaacccgagaggggtgagcgcctacctaagccggcccagcccgttcgacctgttcatccgcaagtcgcccacgatcacctgtctggtggtggacctggcacccagcaaggggaccgtgaacctgacctggtcccgggccagtgggaagcctgtgaaccactccaccagaaaggaggagaagcagcgcaatggcacgttaaccgtcacgtccaccctgccggtgggcacccgagactggatcgagggggagacctaccagtgcagggtgacccacccccacctgcccagggccctcatgcggtccacgaccaagaccagcggcccgcgtgctgccccggaagtctatgcgtttgcgacgccggagtggccggggagccgggacaagcgcaccctcgcctgcctgatccagaacttcatgcctgaggacatctcggtgcagtggctgcacaacgaggtgcagctcccggacgcccggcacagcacgacgcagccccgcaagaccaagggctccggcttcttcgtcttcagccgcctggaggtgaccagggccgaatgggagcagaaagatgagttcatctgccgtgcagtccatgaggcagcgagcccctcacagaccgtccagcgagcggtgtctgtaaatcccggtaaagcggatccttcgaa AA human IgE Fc (CH2—CH3—CH4) ORF: (SEQ ID NO: 543)dhvcsrdftpptvkilqsscdggghfpptiqllclvsgytpgtinitwledgqvmdvdlstasttqegelastqseltlsqkhwlsdrtytcqvtyqghtfedstkkcadsnprgvsaylsrpspfdlfirksptitclvvdlapskgtvnltwsrasgkpvnhstrkeekqrngtltvtstlpvgtrdwiegetyqcrvthphlpralmrsttktsgpraapevyafatpewpgsrdkrtlacliqnfmpedisvqwlhnevqlpdarhsttqprktkgsgffvfsrlevtraeweqkdeficravheaaspsqtvqravsvnpgkadpsIFhIgGwtBcl5 (SEQ ID NO: 544) gtt gtt tga tca gga gcc caa atc ttg tgacaa aac tca cac atg ccc acc gtg ccc agc acc (63 mer) hIgGWT3xba (SEQ IDNO: 545) gtt gtt tct aga tca ttt acc cgg aga cag gga gag gct ctt ctg cgtgta g HuIgGMHWC (sense, 5′ primer for mutating wild type hinge CCC tomutant SSS (SEQ ID NO: 546) gtt gtt gat cag gag ccc aaa tct tct gac aaaact cac aca tct cca ccg tcc cca gca cct gaa ctc ctg ggt gga ccg tca gtcttc c NT 1D8 VH (SEQ ID NO: 547)caggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtccctcacatgcactgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatggatgggaataatatattatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagccaagttttcttaaaaatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaaggagtcatggtcacagtctcctct AA 1D8 VH (no leader) (SEQ ID NO: 548)qvqlkeagpglvqptqtlsltctvsgfsltsdgvhwirqppgkglewmgiiyydggtdynsaiksrlsisrdtsksqvflkinslqtddtamyycarihfdywgqgvmvtvss NT 1D8 VL (no leader) (SEQ IDNO: 549)gacattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgccagctccagtgtaagttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggcttctggagttccaaatcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgccacttattactgtcagcagtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggAA 1D8 VL (SEQ ID NO: 550)divltqspttiaaspgekvtitcrasssvsymywyqqksgaspklwiydtsklasgvpnrfsgsgsgtsyslaintmetedaatyycqqwsstpltfgsgtkleikr NT 1D8 scFv (SEQ ID NO: 551)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgccagctccagtgtaagttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggcttctggagttccaaatcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgccacttattactgtcagcagtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtccctcacatgcactgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatggatgggaataatatattatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagccaagttttcttaaaaatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaaggagtcatggtcacagtctcctctgatca AA 1D8 scFv (SEQ ID NO: 552)mdfqvqifsfllisasvimsrgvdivltqspttiaaspgekvtitcrasssvsymywyqqksgaspklwiydtsklasgvpnrfsgsgsgtsyslaintmetedaatyycqqwsstpltfgsgtkleikrggggsggggsggggsqvqlkeagpglvqptqtlsltctvsgfsltsdgvhwirqppgkglewmgiiyydggtdynsaiksrlsisrdtsksqvflkinslqtddtamyycarihfdywgqgvmvtvss NT 1D8 scFv IgG WTH WTCH2CH3 (SEQ ID NO: 553)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgccagctccagtgtaagttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggcttctggagttccaaatcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgccacttattactgtcagcagtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtccctcacatgcactgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatggatgggaataatatattatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagccaagttttcttaaaaatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaaggagtcatggtcacagtctcctctgatcaggagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga AA 1D8 scFv IgG WTH WTCH2CH3(SEQ ID NO: 554)mdfqvqifsfllisasvimsrgvdivltqspttiaaspgekvtitcrasssvsymywyqqksgaspklwiydtsklasgvpnrfsgsgsgtsyslaintmetedaatyycqqwsstpltfgsgtkleikrggggsggggsggggsqvqlkeagpglvqptqtlsltctvsgfsltsdgvhwirqppgkglewmgiiyydggtdynsaiksrlsisrdtsksqvflkinslqtddtamyycarihfdywgqgvmvtvssdqepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk NT 1D8 scFv IgG MTH MTCH2CH3-CD80 (SEQ ID NO: 555)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgccagctccagtgtaagttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggcttctggagttccaaatcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgccacttattactgtcagcagtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtccctcacatgcactgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatggatgggaataatatattatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagccaagttttcttaaaaatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaaggagtcatggtcacagtctcctctgatctggagcccaaatcttctgacaaaactcacacaagcccaccgagcccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgata AA 1D8 scFv IgG MTH MTCH2CH3-CD80 (SEQ ID NO:556)mdfqvqifsfllisasvimsrgvdivltqspttiaaspgekvtitcrasssvsymywyqqksgaspklwiydtsklasgvpnrfsgsgsgtsyslaintmetedaatyycqqwsstpltfgsgtkleikrggggsggggsggggsqvqlkeagpglvqptqtlsltctvsgfsltsdgvhwirqppgkglewmgiiyydggtdynsaiksrlsisrdtsksqvflkinslqtddtamyycarihfdywgqgvmvtvssdlepkssdkthtsppspapellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkadpsnllpswaitlisvngifviccltycfaprcrerrrnerlrresvrpv NT 1D8 scFv IgGWTH WTCH2CH3-CD80 (SEQ ID NO: 557)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgccagctccagtgtaagttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggcttctggagttccaaatcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgccacttattactgtcagcagtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtccctcacatgcactgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatggatgggaataatatattatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagccaagttttcttaaaaatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaaggagtcatggtcacagtctcctctgatctggagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgata AA 1D8 scFv IgG WTH WTCH2CH3-CD80 (SEQ ID NO:558)mdfqvqifsfllisasvimsrgvdivltqspttiaaspgekvtitcrasssvsymywyqqksgaspklwiydtsklasgvpnrfsgsgsgtsyslaintmetedaatyycqqwsstpltfgsgtkleikrggggsggggsggggsqvqlkeagpglvqptqtlsltctvsgfsltsdgvhwirqppgkglewmgiiyydggtdynsaiksrlsisrdtsksqvflkinslqtddtamyycarihfdywgqgvmvtvssdlepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkadpsnllpswaitlisvngifviccltycfaprcrerrrnerlrresvrpv NT Anti humanCD3 scFv WTH WTCH2CH3 (SEQ ID NO: 559)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattcgcaattatttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctactacacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattgccaacctgcaaccagaagatattgccacttacttttgccaacagggtaatacgcttccgtggacgttcggtggaggcaccaaactggtaaccaaacgggagctcggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctatcgatgaggtccagctgcaacagtctggacctgaactggtgaagcctggagcttcaatgtcctgcaaggcctctggttactcattcactggctacatcgtgaactggctgaagcagagccatggaaagaaccttgagtggattggacttattaatccatacaaaggtcttactacctacaaccagaaattcaagggcaaggccacattaactgtagacaagtcatccagcacagcctacatggagctcctcagtctgacatctgaagactctgcagtctattactgtgcaagatctgggtactatggtgactcggactggtacttcgatgtctggggcgcagggaccacggtcaccgtctcctctgatcaggagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctagaAA Anti human CD3 scFv WTH WTCH2CH3 (SEQ ID NO: 560)Mdfqvqifsfllisasvimsrgvdiqmtqttsslsaslgdrvtiscrasqdirnylnwyqqkpdgtvklliyytsrlhsgvpsrfsgsgsgtdysltianlqpediatyfcqqgntlpwtfgggtklvtkrelggggsggggsggggsidevqlqqsgpelvkpgasmsckasgysftgyivnwlkqshgknlewiglinpykglttynqkfkgkatltvdkssstaymellsltsedsavyycarsgyygdsdwyfdvwgagttvtvssdqepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk NT 2H7-antiCD40 scFv MTH (SSS) MTCH2WTCH3 (SEQ ID NO:561) 2h7-40.2.220Ig + restriction sitesaagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaaggtggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaatccaactctgaagaagcaaagaaagaggaggccaaaaaggaggaagccaagaaatctaacagcgtcgacattgttctgactcagtctccagccaccctgtctgtgactccaggagatagagtctctctttcctgcagggccagccagagtattagcgactacttacactggtatcaacaaaaatcacatgagtctccaaggcttctcatcaaatatgcttcccattccatctctgggatcccctccaggttcagtggcagtggatcagggtcagatttcactctcagtatcaacagtgtggaacctgaagatgttggaatttattactgtcaacatggtcacagctttccgtggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcagatccagttggtgcaatctggacctgagctgaagaagcctggagagacagtcaggatctcctgcaaggcttctgggtatgccttcacaactactggaatgcagtgggtgcaagagatgccaggaaagggtttgaagtggattggctggataaacaccccactctggagtgccaaaatatgtagaagacttcaaggacggtttgccttctctttggaaacctctgccaacactgcatatttacagataagcaacctcaaagatgaggacacggctacgtatttctgtgtgagatccgggaatggtaactatgacctggcctactttgcttactggggccaagggacactggtcactgtctctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga AA 2H7-antiCD40 scFv MTH (SSS) MTCH2WTCH3 (SEQ ID NO: 562)2H7-40.2.220Igmdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkggggsggggsggggssqaylqqsgaelvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvssdqsnseeakkeeakkeeakksnsvdivltqspatlsvtpgdrvslscrasqsisdylhwyqqkshesprllikyashsisgipsrfsgsgsgsdftlsinsvepedvgiyycqhghsfpwtfgggtkleikrggggsggggsggggsqiqlvqsgpelkkpgetvrisckasgyaftttgmqwvqempgkglkwigwintplwsakicrrlqgrfafsletsantaylqisnlkdedtatyfcvrsgngnydlayfaywgqgtlvtvsdqepkssdkthtsppspapellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk NT 5B9 VH (includes the VH leaderpeptide) (SEQ ID NO: 563)atggctgtcttggggctgctcttctgcctggtgacatttccaagctgtgtcctatcccaggtgcagctgaagcagtcaggacctggcctagtgcagtcctcacagagcctgtccatcacctgcacagtctctggtttctcattaactacctatgctgtacactgggttcgccagtctccaggaaagggtctggagtggctgggagtgatatggagtggtggaatcacagactataatgcagctttcatatccagactgagcatcaccaaggacgattccaagagccaagttttctttaaaatgaacagtctgcaacctaatgacacagccatttattactgtgccagaaatgggggtgataactacccttattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctca5B9 VH missing the leader: (SEQ ID NO: 564)caggtgcagctgaagcagtcaggacctggcctagtgcagtcctcacagagcctgtccatcacctgcacagtctctggtttctcattaactacctatgctgtacactgggttcgccagtctccaggaaagggtctggagtggctgggagtgatatggagtggtggaatcacagactataatgcagctttcatatccagactgagcatcaccaaggacgattccaagagccaagttttctttaaaatgaacagtctgcaacctaatgacacagccatttattactgtgccagaaatgggggtgataactacccttattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctca AA 5B9 VH (includes leader peptide) (SEQID NO: 565) MAVLGLLFCLVTFPSCVLSQVQLKQSGPGLVQSSQSLSITCTVSGFSLTTYAVHWVRQSPGKGLEWLGVIWSGGITDYNAAFISRLSITKDDSKSQVFFKMNSLQPNDTAIYYCARNGGDNYPYYYAMDYWGQGTSVTVSS 5B9 VH no leader peptide (SEQ IDNO: 566) QVQLKQSGPGLVQSSQSLSITCTVSGFSLTTYAVHWVRQSPGKGLEWLGVIWSGGITDYNAAFISRLSITKDDSKSQVFFKMNSLQPNDTAIYYCARNGGDNYPYYYAMDYWGQGTSVTVSS NT 5B9 VL (SEQ ID NO: 567)atgaggttctctgctcagcttctggggctgcttgtgctctggatccctggatccactgcagatattgtgatgacgcaggctgcattctccaatccagtcactcttggaacatcagcttccatctcctgcaggtctagtaagagtctcctacatagtaatggcatcacttatttgtattggtatctgcagaagccaggccagtctcctcagctcctgatttatcagatgtccaaccttgcctcaggagtcccagacaggttcagtagcagtgggtcaggaactgatttcacactgagaatcagcagagtggaggctgaggatgtgggtgtttattactgtgctcaaaatctagaacttccgctcacgttcggtgctgggaccaagctggagctgaaacgg AA 5B9 VL (SEQID NO: 568) MRFSAQLLGLLVLWIPGSTADIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPLTFGAGTKLELKR NT 5B9 scFv (SEQ ID NO: 569)aagcttgccgccatgaggttctctgctcagcttctggggctgcttgtgctctggatccctggatccactgcagatattgtgatgacgcaggctgcattctccaatccagtcactcttggaacatcagcttccatctcctgcaggtctagtaagagtctcctacatagtaatggcatcacttatttgtattggtatctgcagaagccaggccagtctcctcagctcctgatttatcagatgtccaaccttgcctcaggagtcccagacaggttcagtagcagtgggtcaggaactgatttcacactgagaatcagcagagtggaggctgaggatgtgggtgtttattactgtgctcaaaatctagaacttccgctcacgttcggtgctgggaccaagctggagctgaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcacaggtgcagctgaagcagtcaggacctggcctagtgcagtcctcacagagcctgtccatcacctgcacagtctctggtttctcattaactacctatgctgtacactgggttcgccagtctccaggaaagggtctggagtggctgggagtgatatggagtggtggaatcacagactataatgcagctttcatatccagactgagcatcaccaaggacgattccaagagccaagttttctttaaaatgaacagtctgcaacctaatgacacagccatttattactgtgccagaaatgggggtgataactacccttattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctct AA 5B9 scFv (SEQ IDNO: 570) MRFSAQLLGLLVLWIPGSTADIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPLTFGAGTKLELKRGGGGSGGGGSGGGGSSQVQLKQSGPGLVQSSQSLSITCTVSGFSLTTYAVHWVRQSPGKGLEWLGVIWSGGITDYNAAFISRLSITKDDSKSQVFFKMNSLQPNDTAIYYCARNGGDNYPYYYAMDYWGQGTSVTV SS NT 5B9scFv-hmtIgG1-hCD80 (SEQ ID NO: 571)aagcttgccgccatgaggttctctgctcagcttctggggctgcttgtgctctggatccctggatccactgcagatattgtgatgacgcaggctgcattctccaatccagtcactcttggaacatcagcttccatctcctgcaggtctagtaagagtctcctacatagtaatggcatcacttatttgtattggtatctgcagaagccaggccagtctcctcagctcctgatttatcagatgtccaaccttgcctcaggagtcccagacaggttcagtagcagtgggtcaggaactgatttcacactgagaatcagcagagtggaggctgaggatgtgggtgtttattactgtgctcaaaatctagaacttccgctcacgttcggtgctgggaccaagctggagctgaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcacaggtgcagctgaagcagtcaggacctggcctagtgcagtcctcacagagcctgtccatcacctgcacagtctctggtttctcattaactacctatgctgtacactgggttcgccagtctccaggaaagggtctggagtggctgggagtgatatggagtggtggaatcacagactataatgcagctttcatatccagactgagcatcaccaaggacgattccaagagccaagttttctttaaaatgaacagtctgcaacctaatgacacagccatttattactgtgccagaaatgggggtgataactacccttattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctctgatctggagcccaaatcttctgacaaaactcacacaagcccaccgagcccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgatactcgag AA5B9 scFv-hmtIgG1-hCD80 (SEQ ID NO: 572)MRFSAQLLGLLVLWIPGSTADIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPLTFGAGTKLELKRGGGGSGGGGSGGGGSSQVQLKQSGPGLVQSSQSLSITCTVSGFSLTTYAVHWVRQSPGKGLEWLGVIWSGGITDYNAAFISRLSITKDDSKSQVFFKMNSLQPNDTAIYYCARNGGDNYPYYYAMDYWGQGTSVTVSSDLEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLR RESVRPV NT2e12 scFv WTH CH2 CH3 (2e12 scFv-WthIgG-CD80) (SEQ ID NO: 573)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgccagagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctcagatctggagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgat 2e12 scFvWTH CH2 CH3 2e12 scFv-WthIgG-CD80 (SEQ ID NO: 574)mdfqvqifsfllisasvimsrgvdivltqspaslavslgqratiscrasesveyyvtslmqwyqqkpgqppkllisaasnvesgvparfsgsgsgtdfslnihpveeddiamyfcqqsrkvpwtfgggtkleikrggggsggggsggggsqvqlkesgpglvapsqslsitctvsgfsltgygvnwvrqppgkglewlgmiwgdgstdynsalksrlsitkdnsksqvflkmnslqtddtaryycardgysnfhyyvmdywgqgtsvtvssdlepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkadpsnllpswaitlisvngifviccltycfaprcrerrrnerlrresvrpvNT 2H7-human IgE Fc (CH2—CH3—CH4) (SEQ ID NO: 575)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaaggtggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctctgatcacgtctgctccagggacttcaccccgcccaccgtgaagatcttacagtcgtcctgcgacggcggcgggcacttccccccgaccatccagctcctgtgcctcgtctctgggtacaccccagggactatcaacatcacctggctggaggacgggcaggtcatggacgtggacttgtccaccgcctctaccacgcaggagggtgagctggcctccacacaaagcgagctcaccctcagccagaagcactggctgtcagaccgcacctacacctgccaggtcacctatcaaggtcacacctttgaggacagcaccaagaagtgtgcagattccaacccgagaggggtgagcgcctacctaagccggcccagcccgttcgacctgttcatccgcaagtcgcccacgatcacctgtctggtggtggacctggcacccagcaaggggaccgtgaacctgacctggtcccgggccagtgggaagcctgtgaaccactccaccagaaaggaggagaagcagcgcaatggcacgttaaccgtcacgtccaccctgccggtgggcacccgagactggatcgagggggagacctaccagtgcagggtgacccacccccacctgcccagggccctcatgcggtccacgaccaagaccagcggcccgcgtgctgccccggaagtctatgcgtttgcgacgccggagtggccggggagccgggacaagcgcaccctcgcctgcctgatccagaacttcatgcctgaggacatctcggtgcagtggctgcacaacgaggtgcagctcccggacgcccggcacagcacgacgcagccccgcaagaccaagggctccggcttcttcgtcttcagccgcctggaggtgaccagggccgaatgggagcagaaagatgagttcatctgccgtgcagtccatgaggcagcgagcccctcacagaccgtccagcgagcggtgtctgtaaatcccggtaaatgataatctaga AA 2H7 scFv IgE (CH2—CH3—CH4) (SEQ ID NO: 576)mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkggggsggggsggggssqaylqqsgaelvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvsdhvcsrdftpptvkilqsscdggghfpptiqllclvsgytpgtinitwledgqvmdvdlstasttqegelastqseltlsqkhwlsdrtytcqvtyqghtfedstkkcadsnprgvsaylsrpspfdlfirksptitclvvdlapskgtvnltwsrasgkpvnhstrkeekqrngtltvtstlpvgtrdwiegetyqcrvthphlpralmrsttktsgpraapevyafatpewpgsrdkrtlacliqnfmpedisvqwlhnevqlpdarhsttqprktkgsgffvfsrlevtraeweqkdeficravheaaspsqtvqravsvnpgk NT 2H7 scFv MH (SSS) MCH2WTCH3 (SEQ ID NO: 577)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctagaAA 2H7 scFv MH (SSS) MCH2WTCH3 (SEQ ID NO: 578)mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggssqaylqqsgaelvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvssdqepkssdkthtsppspapellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk NT 5B9 scFv MTHWTCH2CH3 (SEQ ID NO: 579)aagcttgccgccatgaggttctctgctcagcttctggggctgcttgtgctctggatccctggatccactgcagatattgtgatgacgcaggctgcattctccaatccagtcactcttggaacatcagcttccatctcctgcaggtctagtaagagtctcctacatagtaatggcatcacttatttgtattggtatctgcagaagccaggccagtctcctcagctcctgatttatcagatgtccaaccttgcctcaggagtcccagacaggttcagtagcagtgggtcaggaactgatttcacactgagaatcagcagagtggaggctgaggatgtgggtgtttattactgtgctcaaaatctagaacttccgctcacgttcggtgctgggaccaagctggagctgaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcacaggtgcagctgaagcagtcaggacctggcctagtgcagtcctcacagagcctgtccatcacctgcacagtctctggtttctcattaactacctatgctgtacactgggttcgccagtctccaggaaagggtctggagtggctgggagtgatatggagtggtggaatcacagactataatgcagctttcatatccagactgagcatcaccaaggacgattccaagagccaagttttctttaaaatgaacagtctgcaacctaatgacacagccatttattactgtgccagaaatgggggtgataactacccttattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctagaAA 5B9 scFv MTHWTCH2CH3 (SEQ ID NO: 580)MRFSAQLLGLLVLWIPGSTADIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPLTFGAGTKLELKRGGGGSGGGGSGGGGSSQVQLKQSGPGLVQSSQSLSITCTVSGFSLTTYAVHWVRQSPGKGLEWLGVIWSGGITDYNAAFISRLSITKDDSKSQVFFKMNSLQPNDTAIYYCARNGGDNYPYYYAMDYWGQGTSVTVSSDQEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGKHuman IgG1 hinge mutations 2H7 scFv-MTH (CSS) WTCH2CH3 (SEQ ID NO: 581)Nucleotide:aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttgtgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga2H7 scFv-MTH (CSS) WTCH2CH3 protein: (SEQ ID NO: 582)mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggssqaylqqsgaelvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvssdqepkscdkthtsppspapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk 2H7 scFv-MTH (SCS) WTCH2CH3: (SEQ ID NO: 583)Nucleotide:aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatgcccaccgtccccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga2H7 scFv-MTH (SCS) WTCH2CH3 Protein: (SEQ ID NO: 584)mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggssqaylqqsgaelvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvssdqepkssdkthtcppspapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk 2H7 scFv-MTH (SSC) WTCH2CH3: (SEQ ID NO: 585)Nucleotide:aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga2H7 scFv-MTH (SSC) WTCH2CH3 Protein: (SEQ ID NO: 586)mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggssqaylqqsgaelvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvssdqepkssdkthtsppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk HIgGMHcys1 (SEQ ID NO: 587) gtt gtt gat cag gag cccaaa tct tct gac aaa act cac aca tg HIgGMHcys2 (SEQ ID NO: 588) gtt gttgat cag gag ccc aaa tct tgt gac aaa act cac aca tct cca ccg tgcHIgGMHcys3 (SEQ ID NO: 589) gtt gtt gat cag gag ccc aaa tct tgt gac aaaact cac aca tgt cca ccg tcc cca gca cct NT HuIgG1 MTCH3Y405 (SEQ ID NO:590)gggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttctacctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatgaAA HuIgG1 MTCH3Y405 (SEQ ID NO: 591)GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFYLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK NT HuIgG1MTCH3A405 (SEQ ID NO: 592)gggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcgccctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatga AA HuIgG1 MTCH3A405 (SEQ ID NO: 593)GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK NT HuIgG1MTCH3A407 (SEQ ID NO: 594)Gggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctcgccagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatga AA HuIgG1 MTCH3A407 (SEQ ID NO: 595)GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK NT HuIgG1MTCH3Y405A407 (SEQ ID NO: 596)gggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttctacctcgccagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatga AA HuIgG1 MTCH3Y405A407 (SEQ ID NO: 597)GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFYLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK NT HuIgG1MTCH3A405A407 (SEQ ID NO: 598)gggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcgccctcgccagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatga AA HuIgG1 MTCH3A405A407 (SEQ ID NO: 599)gqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfalaskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk NT 2H7 scFv MTH (SSS)WTCH2MTCH3Y405 (SEQ ID NO: 600)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttctacctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatgatctagaAA 2H7 scFv MTH (SSS) WTCH2MTCH3Y405 (SEQ ID NO: 601)mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggssqaylqqsgaelvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvssdqepkssdkthtsppspapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfylyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk NT 2H7 scFv MTH (SSS) WTCH2MTCH3A405 (SEQ ID NO:602)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcgccctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatgaAA 2H7 scFv MTH (SSS) WTCH2MTCH3A405 (SEQ ID NO: 603)mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggssqaylqqsgaelvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvssdqepkssdkthtsppspapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfalyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk NT 2H7 scFv MTH (SSS) WTCH2MTCH3A407 (SEQ ID NO:604)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctcgccagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatgaAA 2H7 scFv MTH (SSS) WTCH2MTCH3A407 (SEQ ID NO: 605)mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggssqaylqqsgaelvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvssdqepkssdkthtsppspapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflaskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk NT 2H7 scFv MTH (SSS) WTCH2MTCH3Y405A407 (SEQ ID NO:606)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttctacctcgccagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatgaAA 2H7 scFv MTH (SSS) WTCH2MTCH3Y405A407 (SEQ ID NO: 607)mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggssqaylqqsgaelvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvssdqepkssdkthtsppspapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfylaskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk NT 2H7 scFv MTH (SSS) WTCH2MTCH3A405A407 (SEQ ID NO:608)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcgccctcgccagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatgaAA 2H7 scFv MTH (SSS) WTCH2MTCH3A405A407 (SEQ ID NO: 609)mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggssqaylqqsgaelvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvssdqepkssdkthtsppspapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfalaskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk NT 2H7 scFv MTH (SCC) WTCH2CH3 (SEQ ID NO: 610)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctagaAA 2H7 scFv MTH (SCC) WTCH2CH3 (SEQ ID NO: 611)mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggssqaylqqsgaelvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvssdqepkssdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk NT 2H7 scFv MTH (CSC) WTCH2CH3 (SEQ ID NO: 612)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttgtgacaaaactcacacatctccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctagaAA 2H7 scFv MTH (CSC) WTCH2CH3 (SEQ ID NO: 613)mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggssqaylqqsgaelvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvssdqepkscdkthtsppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk NT 2H7 scFv MTH (CCS) WTCH2CH3 (SEQ ID NO: 614)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttgtgacaaaactcacacatgtccaccgtccccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctagaAA 2H7 scFv MTH (CCS) WTCH2CH3 (SEQ ID NO: 615)mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggssqaylqqsgaelvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvssdqepkscdkthtcppspapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk NT HuIgAHIgA-T4-ORF (SEQ ID NO: 616)tgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtcatggcggaggtggacgcggatccttcgaac AA HuIgAHIgA-T4-ORF (SEQ ID NO: 617)Dqpvpstpptpspstpptpspscchprlslhrpaledlllgseailtctltglrdasgvtftwtpssgksavqgppdrdlcgcysvssvlpgcaepwnhgktftctaaypesktpltatlsksgntfrpevhllpppseelalnelvtlclargfspkdvlvrwlqgsqelprekyltwasrqepsqgtttfavtsilrvaaedwkkgdtfscmvghealplaftqktidrlagkpthvnvsvvmaevdadpsn NT HuIgAHIgA-T4-ORF (SEQ ID NO: 618)tgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtcatggcggaggtggacgcggatccttcgaac AA HuIgAHIgA-T4-ORF (SEQ ID NO: 619)dqpvpstpptpspstpptpspscchprlslhrpaledlllgseailtctltglrdasgvtftwtpssgksavqgppdrdlcgcysvssvlpgcaepwnhgktftctaaypesktpltatlsksgntfrpevhllpppseelalnelvtltclargfspkdvlvrwlqgsqelprekyltwasrqepsqgtttfavtsilrvaaedwkkgdtfscmvghealplaftqktidrlagkpthvnvsvvmaevdadpsn NT 1D8-IgAH IgA-T4-CD80 (SEQ ID NO: 620)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgccagctccagtgtaagttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggcttctggagttccaaatcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgccacttattactgtcagcagtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtccctcacatgcactgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatggatgggaataatatattatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagccaagttttcttaaaaatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaaggagtcatggtcacagtctcctctgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtcatggcggaggtggacgcggatccttcgaacaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgatac AA1D8 scFv IgAH IgA-T4-CD80 (SEQ ID NO: 621)mdfqvqifsfllisasvimsrgvdivltqspttiaaspgekvtitcrasssvsymywyqqksgaspklwiydtsklasgvpnrfsgsgsgtsyslaintmetedaatyycqqwsstpltfgsgtkleikrggggsggggsggggsqvqlkeagpglvqptqtlsltctvsgfsltsdgvhwirqppgkglewmgiiyydggtdynsaiksrlsisrdtsksqvflkinslqtddtamyycarihfdywgqgvmvtvssdqpvpstpptpspstpptpspscchprlslhrpaledlllgseailtctltglrdasgvtftwtpssgksavqgppdrdlcgcysvssvlpgcaepwnhgktftctaaypesktpltatlsksgntfrpevhllpppseelalnelvtltclargfspkdvlvrwlqgsqelprekyltwasrqepsqgtttfavtsilrvaaedwkkgdtfscmvghealplaftqktidrlagkpthvnvsvvmaevdadpsnnllpswaitlisvngifviccltycfaprcrerrnerlrresvrpv NT human IgEFc (CH2—CH3—CH4) ORF: (SEQ ID NO: 622)tgatcacgtctgctccagggacttcaccccgcccaccgtgaagatcttacagtcgtcctgcgacggcggcgggcacttccccccgaccatccagctcctgtgcctcgtctctgggtacaccccagggactatcaacatcacctggctggaggacgggcaggtcatggacgtggacttgtccaccgcctctaccacgcaggagggtgagctggcctccacacaaagcgagctcaccctcagccagaagcactggctgtcagaccgcacctacacctgccaggtcacctatcaaggtcacacctttgaggacagcaccaagaagtgtgcagattccaacccgagaggggtgagcgcctacctaagccggcccagcccgttcgacctgttcatccgcaagtcgcccacgatcacctgtctggtggtggacctggcacccagcaaggggaccgtgaacctgacctggtcccgggccagtgggaagcctgtgaaccactccaccagaaaggaggagaagcagcgcaatggcacgttaaccgtcacgtccaccctgccggtgggcacccgagactggatcgagggggagacctaccagtgcagggtgacccacccccacctgcccagggccctcatgcggtccacgaccaagaccagcggcccgcgtgctgccccggaagtctatgcgtttgcgacgccggagtggccggggagccgggacaagcgcaccctcgcctgcctgatccagaacttcatgcctgaggacatctcggtgcagtggctgcacaacgaggtgcagctcccggacgcccggcacagcacgacgcagccccgcaagaccaagggctccggcttcttcgtcttcagccgcctggaggtgaccagggccgaatgggagcagaaagatgagttcatctgccgtgcagtccatgaggcagcgagcccctcacagaccgtccagcgagcggtgtctgtaaatcccggtaaagcggatccttcgaa AA human IgE Fc (CH2—CH3—CH4) ORF: (SEQ ID NO: 623)dhvcsrdftpptvkilqsscdggghfpptiqllclvsgytpgtinitwledgqvmdvdlstasttqegelastqseltlsqkhwlsdrtytcqvtyqghtfedstkkcadsnprgvsaylsrpspfdlfirksptitclvvdlapskgtvnltwsrasgkpvnhstrkeekqrngtltvtstlpvgtrdwiegetyqcrvthphlpralmrsttktsgpraapevyafatpewpgsrdkrtlacliqnfmpedisvqwlhnevqlpdarhsttqprktkgsgffvfsrlevtraeweqkdeficravheaaspsqtvqravsvnpgkadpsNT 1D8 scFv-human IgE Fc (CH2—CH3—CH4)-CD80 (SEQ ID NO: 624)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgccagctccagtgtaagttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggcttctggagttccaaatcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgccacttattactgtcagcagtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtccctcacatgcactgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatggatgggaataatatattatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagccaagttttcttaaaaatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaaggagtcatggtcacagtctcctctgatcacgtctgctccagggacttcaccccgcccaccgtgaagatcttacagtcgtcctgcgacggcggcgggcacttccccccgaccatccagctcctgtgcctcgtctctgggtacaccccagggactatcaacatcacctggctggaggacgggcaggtcatggacgtggacttgtccaccgcctctaccacgcaggagggtgagctggcctccacacaaagcgagctcaccctcagccagaagcactggctgtcagaccgcacctacacctgccaggtcacctatcaaggtcacacctttgaggacagcaccaagaagtgtgcagattccaacccgagaggggtgagcgcctacctaagccggcccagcccgttcgacctgttcatccgcaagtcgcccacgatcacctgtctggtggtggacctggcacccagcaaggggaccgtgaacctgacctggtcccgggccagtgggaagcctgtgaaccactccaccagaaaggaggagaagcagcgcaatggcacgttaaccgtcacgtccaccctgccggtgggcacccgagactggatcgagggggagacctaccagtgcagggtgacccacccccacctgcccagggccctcatgcggtccacgaccaagaccagcggcccgcgtgctgccccggaagtctatgcgtttgcgacgccggagtggccggggagccgggacaagcgcaccctcgcctgcctgatccagaacttcatgcctgaggacatctcggtgcagtggctgcacaacgaggtgcagctcccggacgcccggcacagcacgacgcagccccgcaagaccaagggctccggcttcttcgtcttcagccgcctggaggtgaccagggccgaatgggagcagaaagatgagttcatctgccgtgcagtccatgaggcagcgagcccctcacagaccgtccagcgagcggtgtctgtaaatcccggtaaagcggatccttcgaagctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgata AA 1D8-scFv-human IgEFc (CH2—CH3—CH4)-CD80 (SEQ ID NO: 625)mdfqvqifsfllisasvimsrgvdivltqspttiaaspgekvtitcrasssvsymywyqqksgaspklwiydtsklasgvpnrfsgsgsgtsyslaintmetedaatyycqqwsstpltfgsgtkleikrggggsggggsggggsqvqlkeagpglvqptqtlsltctvsgfsltsdgvhwirqppgkglewmgiiyydggtdynsaiksrlsisrdtsksqvflkinslqtddtamyycarihfdywgqgvmvtvssdhvcsrdftpptvkilqsscdggghfpptiqllclvsgytpgtinitwledgqvmdvdlstasttqegelastqseltlsqkhwlsdrtytcqvtyqghtfedstkkcadsnprgvsaylsrpspfdlfirksptitclvvdlapskgtvnltwsrasgkpvnhstrkeekqrngtltvtstlpvgtrdwiegetyqcrvthphlpralmrsttktsgpraapevyafatpewpgsrdkrtlacliqnfmpedisvqwlhnevqlpdarhsttqprktkgsgffvfsrlevtraeweqkdeficravheaaspsqtvqravsvnpgkadpsklpswaitlisvngifviccltycfaprcrerrrnerlrresvrpv NT 5B9-IgAHIgA-T4-CD80 (SEQ ID NO: 626)aagcttgccgccatgaggttctctgctcagcttctggggctgcttgtgctctggatccctggatccactgcagatattgtgatgacgcaggctgcattctccaatccagtcactcttggaacatcagcttccatctcctgcaggtctagtaagagtctcctacatagtaatggcatcacttatttgtattggtatctgcagaagccaggccagtctcctcagctcctgatttatcagatgtccaaccttgcctcaggagtcccagacaggttcagtagcagtgggtcaggaactgatttcacactgagaatcagcagagtggaggctgaggatgtgggtgtttattactgtgctcaaaatctagaacttccgctcacgttcggtgctgggaccaagctggagctgaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcacaggtgcagctgaagcagtcaggacctggcctagtgcagtcctcacagagcctgtccatcacctgcacagtctctggtttctcattaactacctatgctgtacactgggttcgccagtctccaggaaagggtctggagtggctgggagtgatatggagtggtggaatcacagactataatgcagctttcatatccagactgagcatcaccaaggacgattccaagagccaagttttctttaaaatgaacagtctgcaacctaatgacacagccatttattactgtgccagaaatgggggtgataactacccttattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctctgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtcatggcggaggtggacgcggatccttcgaacaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgatac AA 5B9-IgAH IgA-T4-CD80 (SEQ ID NO: 627)mrfsaqllgllvlwipgstadivmtqaafsnpvtlgtsasiscrssksllhsngitylywylqkpgqspqlliyqmsnlasgvpdrfsssgsgtdftlrisrveaedvgvyycaqnlelpltfgagtklelkrggggsggggsggggssqvqlkqsgpglvqssqslsitctvsgfslttyavhwvrqspgkglewlgviwsggitdynaafisrlsitkddsksqvffkmnslqpndtaiyycarnggdnypyyyamdywgqgtsvtvssdqpvpstpptpspstpptpspscchprlslhrpaledlllgseailtctltglrdasgvtftwtpssgksavqgppdrdlcgcysvssvlpgcaepwnhgktftctaaypesktpltatlsksgntfrpevhllpppseelalnelvtltclargfspkdvlvrwlqgsqelprekyltwasrqepsqgtttfavtsilrvaaedwkkgdtfscmvghealplaftqktidrlagkpthvnvsvvmaevdadpsnnllpswaitlisvngifviccltycfaprcrerrrnerlrresvrpvNT 5B9-scFv-human IgE Fc (CH2—CH3—CH4)-CD80 (SEQ ID NO: 628)aagcttgccgccatgaggttctctgctcagcttctggggctgcttgtgctctggatccctggatccactgcagatattgtgatgacgcaggctgcattctccaatccagtcactcttggaacatcagcttccatctcctgcaggtctagtaagagtctcctacatagtaatggcatcacttatttgtattggtatctgcagaagccaggccagtctcctcagctcctgatttatcagatgtccaaccttgcctcaggagtcccagacaggttcagtagcagtgggtcaggaactgatttcacactgagaatcagcagagtggaggctgaggatgtgggtgtttattactgtgctcaaaatctagaacttccgctcacgttcggtgctgggaccaagctggagctgaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcacaggtgcagctgaagcagtcaggacctggcctagtgcagtcctcacagagcctgtccatcacctgcacagtctctggtttctcattaactacctatgctgtacactgggttcgccagtctccaggaaagggtctggagtggctgggagtgatatggagtggtggaatcacagactataatgcagctttcatatccagactgagcatcaccaaggacgattccaagagccaagttttctttaaaatgaacagtctgcaacctaatgacacagccatttattactgtgccagaaatgggggtgataactacccttattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctctgatcacgtctgctccagggacttcaccccgcccaccgtgaagatcttacagtcgtcctgcgacggcggcgggcacttccccccgaccatccagctcctgtgcctcgtctctgggtacaccccagggactatcaacatcacctggctggaggacgggcaggtcatggacgtggacttgtccaccgcctctaccacgcaggagggtgagctggcctccacacaaagcgagctcaccctcagccagaagcactggctgtcagaccgcacctacacctgccaggtcacctatcaaggtcacacctttgaggacagcaccaagaagtgtgcagattccaacccgagaggggtgagcgcctacctaagccggcccagcccgttcgacctgttcatccgcaagtcgcccacgatcacctgtctggtggtggacctggcacccagcaaggggaccgtgaacctgacctggtcccgggccagtgggaagcctgtgaaccactccaccagaaaggaggagaagcagcgcaatggcacgttaaccgtcacgtccaccctgccggtgggcacccgagactggatcgagggggagacctaccagtgcagggtgacccacccccacctgcccagggccctcatgcggtccacgaccaagaccagcggcccgcgtgctgccccggaagtctatgcgtttgcgacgccggagtggccggggagccgggacaagcgcaccctcgcctgcctgatccagaacttcatgcctgaggacatctcggtgcagtggctgcacaacgaggtgcagctcccggacgcccggcacagcacgacgcagccccgcaagaccaagggctccggcttcttcgtcttcagccgcctggaggtgaccagggccgaatgggagcagaaagatgagttcatctgccgtgcagtccatgaggcagcgagcccctcacagaccgtccagcgagcggtgtctgtaaatcccggtaaagcggatccttcgaagctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgata AA 5B9-scFv-human IgE Fc (CH2—CH3—CH4)-CD80 (SEQ ID NO: 629)mrfsaqllgllvlwipgstadivmtqaafsnpvtlgtsasiscrssksllhsngitylywylqkpgqspqlliyqmsnlasgvpdrfsssgsgtdftlrisrveaedvgvyycaqnlelpltfgagtklelkrggggsggggsggggssqvqlkqsgpglvqssqslsitctvsgfslttyavhwvrqspgkglewlgviwsggitdynaafisrlsitkddsksqvffkmnslqpndtaiyycarnggdnypyyyamdywgqgtsvtvssdhvcsrdftpptvkilqsscdggghfpptiqllclvsgytpgtinitwledgqvmdvdlstasttqegelastqseltlsqkhwlsdrtytcqvtyqghtfedstkkcadsnprgvsaylsrpspfdlfirksptitclvvdlapskgtvnltwsrasgkpvnhstrkeekqrngtltvtstlpvgtrdwiegetyqcrvthphlpralmrsttktsgpraapevyafatpewpgsrdkrtlacliqnfmpedisvqwlhnevqlpdarhsttqprktkgsgffvfsrlevtraeweqkdeficravheaaspsqtvqravsvnpgkadpsklpswaitlisvngifviccltycfaprcrerrrnerlrresvrpv NT2e12-scFv-IgAH IgA-T4-CD80 (SEQ ID NO: 630)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgccagagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctcagatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtcatggcggaggtggacgcggatccttcgaacaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgatac AA 2e12-scFv-IgAH IgA-T4-CD80 (SEQ ID NO:631)mdfqvqifsfllisasvimsrgvdivltqspaslavslgqratiscrasesveyyvtslmqwyqqkpgqppkllisaasnvesgvparfsgsgsgtdfslnihpveeddiamyfcqqsrkvpwtfgggtkleikrggggsggggsggggsqvqlkesgpglvapsqslsitctvsgfsltgygvnwvrqppgkglewlgmiwgdgstdynsalksrlsitkdnsksqvflkmnslqtddtaryycardgysnfhyyvmdywgqgtsvtvssdqpvpstpptpspstpptpspscchprlslhrpaledlllgseailtctltglrdasgvtftwtpssgksavqgppdrdlcgcysvssvlpgcaepwnhgktftctaaypesktpltatlsksgntfrpevhllpppseelalnelvtltclargfspkdvlvrwlqgsqelprekyltwasrqepsqgtttfavtsilrvaaedwkkgdtfscmvghealplaftqktidrlagkpthvnvsvvmaevdadpsnnllpswaitlisvngifviccltycfaprcrerrrnerlrresvrpvNT 2e12-scFv-human IgE Fc (CH2—CH3—CH4)-CD80 (SEQ ID NO: 632)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgccagagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctcagatcacgtctgctccagggacttcaccccgcccaccgtgaagatcttacagtcgtcctgcgacggcggcgggcacttccccccgaccatccagctcctgtgcctcgtctctgggtacaccccagggactatcaacatcacctggctggaggacgggcaggtcatggacgtggacttgtccaccgcctctaccacgcaggagggtgagctggcctccacacaaagcgagctcaccctcagccagaagcactggctgtcagaccgcacctacacctgccaggtcacctatcaaggtcacacctttgaggacagcaccaagaagtgtgcagattccaacccgagaggggtgagcgcctacctaagccggcccagcccgttcgacctgttcatccgcaagtcgcccacgatcacctgtctggtggtggacctggcacccagcaaggggaccgtgaacctgacctggtcccgggccagtgggaagcctgtgaaccactccaccagaaaggaggagaagcagcgcaatggcacgttaaccgtcacgtccaccctgccggtgggcacccgagactggatcgagggggagacctaccagtgcagggtgacccacccccacctgcccagggccctcatgcggtccacgaccaagaccagcggcccgcgtgctgccccggaagtctatgcgtttgcgacgccggagtggccggggagccgggacaagcgcaccctcgcctgcctgatccagaacttcatgcctgaggacatctcggtgcagtggctgcacaacgaggtgcagctcccggacgcccggcacagcacgacgcagccccgcaagaccaagggctccggcttcttcgtcttcagccgcctggaggtgaccagggccgaatgggagcagaaagatgagttcatctgccgtgcagtccatgaggcagcgagcccctcacagaccgtccagcgagcggtgtctgtaaatcccggtaaagcggatccttcgaagctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgata AA 2e12-scFv-human IgE Fc (CH2—CH3—CH4)-CD80 (SEQ ID NO: 633)mdfqvqifsfllisasvimsrgvdivltqspaslavslgqratiscrasesveyyvtslmqwyqqkpgqppkllisaasnvesgvparfsgsgsgtdfslnihpveeddiamyfcqqsrkvpwtfgggtkleikrggggsggggsggggsqvqlkesgpglvapsqslsitctvsgfsltgygvnwvrqppgkglewlgmiwgdgstdynsalksrlsitkdnsksqvflkmnslqtddtaryycardgysnfhyyvmdywgqgtsvtvssdhvcsrdftpptvkilqsscdggghfpptiqllclvsgytpgtinitwledgqvmdvdlstasttqegelastqseltlsqkhwlsdrtytcqvtyqghtfedstkkcadsnprgvsaylsrpspfdlfirksptitclvvdlapskgtvnltwsrasgkpvnhstrkeekqrngtltvtstlpvgtrdwiegetyqcrvthphlpralmrsttktsgpraapevyafatpewpgsrdkrtlacliqnfmpedisvqwlhnevqlpdarhsttqprktkgsgffvfsrlevtraeweqkdeficravheaaspsqtvqravsvnpgkadpsklpswaitlisvngifviccltycfaprcrerrrnerlrresvrpv NT500A2 scFv (SEQ ID NO: 634)atgttgtatacatctcagctccttgggcttttactcttctggatttcagcctccagaagtgacatagtgctgactcagactccagccactctgtctctaattcctggagaaagagtcacaatgacctgtaagaccagtcagaatattggcacaatcttacactggtatcaccaaaaaccaaaggaggctccaagggctctcatcaagtatgcttcgcagtccattcctgggatcccctccagattcagtggcagtggttcggaaacagatttcactctcagcatcaataacctggagcctgatgatatcggaatttattactgtcaacaaagtagaagctggcctgtcacgttcggtcctggcaccaagctggagataaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcaggtcaagctgcagcagtccggttctgaactagggaaacctggggcctcagtgaaactgtcctgcaagacttcaggctacatattcacagatcactatatttcttgggtgaaacagaagcctggagaaagcctgcagtggataggaaatgtttatggtggaaatggtggtacaagctacaatcaaaaattccagggcaaggccacactgactgtagataaaatctctagcacagcctacatggaactcagcagcctgacatctgaggattctgccatctattactgtgcaagaaggccggtagcgacgggccatgctatggactactggggtcaggggatccaagttaccgtctcctctgatc AA 500A2 scFv (SEQ ID NO: 635)mlytsqllglllfwisasrsdivltqtpatlslipgervtmtcktsqnigtilhwyhqkpkeapralikyasqsipgipsrfsgsgsetdftlsinnlepddigiyycqqsrswpvtfgpgtkleikrggggsggggsggggsqvklqqsgselgkpgasvklscktsgyiftdhyiswvkqkpgeslqwignvyggnggtsynqkfqgkatltvdkisstaymelssltsedsaiyycarrpvatghamdywgqgiqvtvssd 5′ oligo: Name: hIgAbel5 (SEQ ID NO: 636)Sequence: GTTGTTGATCAGCCAGTTCCCTCAACTCCACCTACC 3′ oligo: Name: IgA3BB(SEQ ID NO: 637) GTTGTTTTCGAAGGATCCGCGTCCACCTCCGCCATGACAACAGA 5′ oligo:Name: IgGWT3 (SEQ ID NO: 638)GTTGTTTTCGAAGGATCCGCTTTACCCGGAGACAGGGAGAGGCT CTT 3′ oligo: Name: hIgGWT5(SEQ ID NO: 639) GTTGTTAGATCTGGAGCCCAAATCTTGTGACAAAACTCACACATG 5′ oligo:Name: FADD5 (SEQ ID NO: 640) Sequence:GTTGTGGATCCTTCGAACCCGTTCCTGGTGCTGCTGCACTCGGTGTCG 3′ oligo: Name: FADD3(SEQ ID NO: 641) Sequence:GTTGTTATCGATCTCGAGTTATCAGGACGCTTCGGAGGTAGATGCGTC FADD-CSSCFV: (SEQ IDNO: 642)GtggatccttcgaacccgttcctggtgctgctgcactcggtgtcgtccagcctgtcgagcagcgagctgaccgagctcaagttcctatgcctcgggcgcgtgggcaagcgcaagctggagcgcgtgcagagcggcctagacctcttctccatgctgctggagcagaacgacctggagcccgggcacaccgagctcctgcgcgagctgctcgcctccctgcggcgccacgacctgctgcggcgcgtcgacgacttcgaggcgggggcggcggccggggccgcgcctggggaagaagacctgtgtgcagcatttaacgtcatatgtgataatgtggggaaagattggagaaggctggctcgtcagctcaaagtctcagacaccaagatcgacagcatcgaggacagatacccccgcaacctgacagagcgtgtgcgggagtcactgagaatctggaagaacacagagaaggagaacgcaacagtggcccacctggtgggggctctcaggtcctgccagatgaacctggtggctgacctggtacaagaggttcagcaggcccgtgacctccagaacaggagtggggccatgtccccgatgtcatggaactcagacgcatctacctccgaagcgtcctgataactcgagatcgataacaacPeptide sequence: (SEQ ID NO: 643)vdpsnpflvllhsvssslssseltelkflclgrvgkrklervqsgldlfsmlleqndlepghtellrellaslrrhdllrrvddfeagaaagaapgeedlcaafnvicdnvgkdwrrlarqlkvsdtkidsiedryprnltervreslriwkntekenatvahlvgalrscqmnlvadlvqevqqardlqnrsgamspmswnsdastseas Name: HCD28tm5B (SEQID NO: 644) GTTGTGGATCCTCCCTTTTGGGTGCTGGTGGTGGTTGGTGTCCTGGCTTGCTATAGCTTG Name: HCD28tm3S (SEQ ID NO: 645)GTTGTTTCGAACCCAGAAAATAATAAAGGCCACTGTTACTAGCA AGCTATAGCAAGCCAG HCD28tm5′(SEQ ID NO: 646) GTTGTGGATCCTCCCTTTTGGGTGCTGGTGGT HCD28tm3′ (SEQ ID NO:647) GTTGTTTCGAACCCAGAAAATAATAAAGGCCAC HCD80tm5′ (SEQ ID NO: 648)GTTGTGGATCCTCCTGCTCCCATCCTGG HCD80tm3′ (SEQ ID NO: 649)GTTGTTTCGAACGGCAAAGCAGTAGGTCAGGC Name: MFADD5BB (SEQ ID NO: 650)Sequence: GTTGTGGATCCTTCGAACCCATTCCTGGTGCTGCTGCACTCGCTG Name: MFADD3XC(SEQ ID NO: 651) Sequence: GTTGTTATCGATCTCGAGTCAGGGTGTTTCTGAGGAAGACACMurine FADD Nucleotide sequence (full length, but without flanking-Ig ortransmembrane sequences): (SEQ ID NO: 652)GtggatccttcgaacatggacccattcctggtgctgctgcactcgctgtccggcagcctgtcgggcaacgatctgatggagctcaagttcttgtgccgcgagcgcgtgagcaaacgaaagctggagcgcgtgcagagtggcctggacctgttcacggtgctgctggagcagaacgacctggagcgcgggcacaccgggctgctgcgcgagttgctggcctcgctgcgccgacacgatctactgcagcgcctggacgacttcgaggcggggacggcgaccgctgcgcccccgggggaggcagatctgcaggtggcatttgacattgtgtgtgacaatgtggggagagactggaaaagactggcccgcgagctgaaggtgtctgaggccaagatggatgggattgaggagaagtacccccgaagtctgagtgagcgggtaagggagagtctgaaagtctggaagaatgctgagaagaagaacgcctcggtggccggactggtcaaggcgctgcggacctgcaggctgaatctggtggctgacctggtggaagaagcccaggaatctgtgagcaagagtgagaatatgtccccagtactaagggattcaactgtgtcttcctcagaaacaccctgactcgagatcgatMurine FADD (SEQ ID NO: 653)vdpsnmdpflvllhslsgslsgndlmelkflcrervskrklervqsgldlftvlleqndlerghtgllrellaslrrhdllqrlddfeagtataappgeadlqvafdivcdnvgrdwkrlarelkvseakmdgieekyprslservreslkvwknaekknasvaglvkalrtcrlnlvadlveeaqesvsksenmspvlrdstvsssetp Name: MCASP3-5 (SEQID NO: 654) Sequence: GTTGTGGATCCTTCGAACATGGAGAACAACAAAACCTCAGTGGATTCAName: MCASP3-3 (SEQ ID NO: 655) Sequence:GTTGTTATCGATCTCGAGCTAGTGATAAAAGTACAGTTCTTTCGT Name: mcasp8-5 (SEQ ID NO:656) Sequence: GTTGTTTCGAACATGGATTTCCAGAGTTGTCTTTATGCTATTGCTG Name:mcasp8-3 (SEQ ID NO: 657) Sequence:GTTGTTATCGATCTCGAGTCATTAGGGAGGGAAGAAGAGCTTCTTCCG Name: hcasp3-5 (SEQ IDNO: 658) Sequence: GTTGTGGATCCTTCGAACATGGAGAACACTGAAAACTCAGTGGAT Name:hcasp3-3 (SEQ ID NO: 659) Sequence:GTTGTTATCGATCTCGAGTTAGTGATAAAAATAGAGTTCTTTTGTGAG Name: hcasp8-5 (SEQ IDNO: 660) Sequence: GTTGTGGATCCTTCGAACATGGACTTCAGCAGAAATCTTTATGAT Name:hcasp8-3 (SEQ ID NO: 661) Sequence:GTTGTTATCGATGCATGCTCAATCAGAAGGGAAGACAAGTTTTTTTCT HuIgGMHWC (SEQ ID NO:662) gtt gtt gat cag gag ccc aaa tct tct gac aaa act cac aca tct cca ccgtcc cca gca cct gaa ctc ctg ggt gga ccg tca gtc ttc c NT 2H7-human IgE(CH2—CH3—CH4) (SEQ ID NO: 663)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaaggtggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctctgatcacgtctgctccagggacttcaccccgcccaccgtgaagatcttacagtcgtcctgcgacggcggcgggcacttccccccgaccatccagctcctgtgcctcgtctctgggtacaccccagggactatcaacatcacctggctggaggacgggcaggtcatggacgtggacttgtccaccgcctctaccacgcaggagggtgagctggcctccacacaaagcgagctcaccctcagccagaagcactggctgtcagaccgcacctacacctgccaggtcacctatcaaggtcacacctttgaggacagcaccaagaagtgtgcagattccaacccgagaggggtgagcgcctacctaagccggcccagcccgttcgacctgttcatccgcaagtcgcccacgatcacctgtctggtggtggacctggcacccagcaaggggaccgtgaacctgacctggtcccgggccagtgggaagcctgtgaaccactccaccagaaaggaggagaagcagcgcaatggcacgttaaccgtcacgtccaccctgccggtgggcacccgagactggatcgagggggagacctaccagtgcagggtgacccacccccacctgcccagggccctcatgcggtccacgaccaagaccagcggcccgcgtgctgccccggaagtctatgcgtttgcgacgccggagtggccggggagccgggacaagcgcaccctcgcctgcctgatccagaacttcatgcctgaggacatctcggtgcagtggctgcacaacgaggtgcagctcccggacgcccggcacagcacgacgcagccccgcaagaccaagggctccggcttcttcgtcttcagccgcctggaggtgaccagggccgaatgggagcagaaagatgagttcatctgccgtgcagtccatgaggcagcgagcccctcacagaccgtccagcgagcggtgtctgtaaatcccggtaaatgataatctaga AA 2H7 scFv IgE (CH2—CH3—CH4) (SEQ ID NO: 664)mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkggggsggggsggggssqaylqqsgaelvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvsdhvcsrdftpptvkilqsscdggghfpptiqllclvsgytpgtinitwledgqvmdvdlstasttqegelastqseltlsqkhwlsdrtytcqvtyqghtfedstkkcadsnprgvsaylsrpspfdlfirksptitclvvdlapskgtvnltwsrasgkpvnhstrkeekqrngtltvtstlpvgtrdwiegetyqcrvthphlpralmrsttktsgpraapevyafatpewpgsrdkrtlacliqnfmpedisvqwlhnevqlpdarhsttqprktkgsgffvfsrlevtraeweqkdeficravheaaspsqtvqravsvnpgk NT 2H7 scFv MH (SSS) MCH2WTCH3 (SEQ ID NO: 665)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctagaAA 2H7 scFv MH (SSS) MCH2WTCH3 (SEQ ID NO: 666)mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggssqaylqqsgaelvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvssdqepkssdkthtsppspapellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk NT 5B9 scFv MTHWTCH2CH3 (SEQ ID NO: 667)aagcttgccgccatgaggttctctgctcagcttctggggctgcttgtgctctggatccctggatccactgcagatattgtgatgacgcaggctgcattctccaatccagtcactcttggaacatcagcttccatctcctgcaggtctagtaagagtctcctacatagtaatggcatcacttatttgtattggtatctgcagaagccaggccagtctcctcagctcctgatttatcagatgtccaaccttgcctcaggagtcccagacaggttcagtagcagtgggtcaggaactgatttcacactgagaatcagcagagtggaggctgaggatgtgggtgtttattactgtgctcaaaatctagaacttccgctcacgttcggtgctgggaccaagctggagctgaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcacaggtgcagctgaagcagtcaggacctggcctagtgcagtcctcacagagcctgtccatcacctgcacagtctctggtttctcattaactacctatgctgtacactgggttcgccagtctccaggaaagggtctggagtggctgggagtgatatggagtggtggaatcacagactataatgcagctttcatatccagactgagcatcaccaaggacgattccaagagccaagttttctttaaaatgaacagtctgcaacctaatgacacagccatttattactgtgccagaaatgggggtgataactacccttattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctagaAA 5B9 scFv MTHWTCH2CH3 (SEQ ID NO: 668)mrfsaqllgllvlwipgstadivmtqaafsnpvtlgtsasiscrssksllhsngitylywylqkpgqspqlliyqmsnlasgvpdrfsssgsgtdftlrisrveaedvgvyycaqnlelphfgagtklelkrggggsggggsggggssqvqlkqsgpglvqssqslsitctvsgfslttyavhwvrqspgkglewlgviwsggitdynaafisrlsitkddsksqvffkmnslqpndtaiyycarnggdnypyyyamdywgqgtsvtvssdqepkssdkthtsppspapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for the purposeof illustration, various modifications may be made without deviatingfrom the spirit and scope of the invention. Accordingly, the presentinvention is not limited except as by the appended claims.

1.-61. (canceled)
 62. A method for treating a disease involving B cellactivity, comprising administering to a subject in need thereof atherapeutically effective amount of a fusion protein that comprises: (a)a binding domain polypeptide that binds CD20, wherein the binding domaincomprises amino acids 23-265 of SEQ ID NO:397 except that leucine atposition 155 of SEQ ID NO:397 is substituted with a serine, (b) a humanIgG1 hinge peptide comprising a mutated core sequence as set forth inamino acids 279-282 of SEQ ID NO:397, 398 or 582, (c) an immunoglobulinheavy chain CH2 constant region polypeptide as set forth in amino acids284-393 of SEQ ID NO:397 fused to the hinge peptide, and (d) animmunoglobulin heavy chain CH3 constant region polypeptide as set forthin amino acids 394-500 of SEQ ID NO:397 fused to the CH2 polypeptide,wherein the fusion protein promotes antibody dependent cell-mediatedcytotoxicity, complement fixation, or both.
 63. The method of claim 62,wherein the disease involving B cell activity is psoriasis, systemiclupus erythematosus, type 1 diabetes mellitus, multiple sclerosis,inflammatory bowel disease, Crohn's disease, or ulcerative colitis. 64.The method of claim 62, wherein the disease is rheumatoid arthritis. 65.The method of claim 62, wherein the human IgG1 hinge peptide of thefusion protein comprises amino acids 279-282 as set forth in SEQ IDNO:397.
 66. The method of claim 62, wherein the human IgG1 hinge peptideof the fusion protein comprises amino acids 279-282 as set forth in SEQID NO:398.
 67. The method of claim 62, wherein the human IgG1 hingepeptide of the fusion protein comprises amino acids 279-282 as set forthin SEQ ID NO:582.
 68. The method of claim 63, wherein the human IgG1hinge peptide of the fusion protein comprises amino acids 279-282 as setforth in SEQ ID NO:397.
 69. The method of claim 63, wherein the humanIgG1 hinge peptide of the fusion protein comprises amino acids 279-282as set forth in SEQ ID NO:398.
 70. The method of claim 63, wherein thehuman IgG1 hinge peptide of the fusion protein comprises amino acids279-282 as set forth in SEQ ID NO:582.
 71. The method of claim 64,wherein the human IgG1 hinge peptide of the fusion protein comprisesamino acids 279-282 as set forth in SEQ ID NO:397.
 72. The method ofclaim 64, wherein the human IgG1 hinge peptide of the fusion proteincomprises amino acids 279-282 as set forth in SEQ ID NO:398.
 73. Themethod of claim 64, wherein the human IgG1 hinge peptide of the fusionprotein comprises amino acids 279-282 as set forth in SEQ ID NO:582. 74.A method for treating a disease involving B cell activity, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a fusion protein that comprises: (a) a binding domainpolypeptide that binds CD20, wherein the binding domain comprisescomplementary determining regions of amino acids 46-55, 71-76, and110-118 of an immunoglobulin light chain variable region as set forth inamino acids 23-128 of SEQ ID NO:397 fused via a linker polypeptide to asequence comprising complementary determining regions of amino acids175-179, 194-210, and 243-255 of a full-length immunoglobulin heavychain variable region as set forth in amino acids 145-265 of SEQ IDNO:397, wherein the linker comprises amino acids 129-144 of SEQ IDNO:397, (b) a human IgG1 hinge peptide comprising a mutated coresequence as set forth in amino acids 279-282 of SEQ ID NO:397, (c) animmunoglobulin heavy chain CH2 constant region polypeptide as set forthin amino acids 284-393 of SEQ ID NO:397 fused to the hinge peptide, and(d) an immunoglobulin heavy chain CH3 constant region polypeptide as setforth in amino acids 394-500 of SEQ ID NO:397 fused to the CH2polypeptide, wherein the fusion protein promotes antibody dependentcell-mediated cytotoxicity, complement fixation, or both.
 75. The methodof claim 74, wherein the disease involving B cell activity is psoriasis,systemic lupus erythematosus, type 1 diabetes mellitus, multiplesclerosis, inflammatory bowel disease, Crohn's disease, or ulcerativecolitis.
 76. The method of claim 74, wherein the disease is rheumatoidarthritis.
 77. The method of claim 74, wherein the binding domainpolypeptide has a serine at position 11 in the immunoglobulin heavychain variable region polypeptide of the fusion protein.
 78. The methodof claim 75, wherein the binding domain polypeptide has a serine atposition 11 in the immunoglobulin heavy chain variable regionpolypeptide of the fusion protein.
 79. The method of claim 76, whereinthe binding domain polypeptide has a serine at position 11 in theimmunoglobulin heavy chain variable region polypeptide of the fusionprotein.